Modified tumor necrosis factor-alpha converting enzyme and methods of use thereof

ABSTRACT

The present invention discloses a modified tumor necrosis factor-alpha converting enzyme (TACE) catalytic domain, that unlike the native TACE catalytic domain, is stable at high protein concentrations. The present invention further discloses methods for generating crystals of the modified TACE protein in protein-ligand complexes with a number of inhibitors. In addition, the present invention discloses methods of using the proteins, crystals and/or three-dimensional structures obtained to identify compounds that can modulate the enzymatic activity of TACE.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional application that claims priorityunder 35 U.S.C. § 119(e) of provisional application, U.S. Ser. No.60/383,391 filed May 24, 2002, the contents of which are herebyincorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention pertains to a modified tumor necrosis factor-alphaconverting enzyme (TACE). The present invention further pertains togenerating a crystal of the modified TACE protein in protein-ligandcomplex with a selected inhibitor for use in structure based rationaldrug design. In addition, the present invention pertains to methods ofusing these proteins and crystals to identify compounds that canmodulate the enzymatic activity of TACE.

2. Background

Tumor necrosis factor-alpha (TNF-alpha) plays a major role in the immuneand inflammatory responses in mammals [Qi and Pekala, Proc Soc Exp BiolMed. 223(2):128–135 (2000)]. First isolated from the serum of rabbitsthat had been treated with endotoxin, TNF-alpha was named for itsability to trigger the hemorrhagic necrosis of specific transplantabletumors [Old, Science 230:630-633 (1985)]. Subsequently, TNF-alpha wasfound to be identical to cachetin, a protein that is intimately involvedin cachexia, the wasting disease prevalent in AIDS and cancer [Beutleret al., J. Exp. Med., 161:984-995 (1985)].

TNF-alpha can bind to two distinct cell membrane receptors (TNFR1 andTNFR2) to transduce intercellular signals to a variety of differenttarget cells in a number of different tissues [Qi and Pekala, Proc SocExp Biol Med. 223(2):128-135 (2000)]. Though a central participant inseveral key cellular processes, TNF-alpha also functions deleteriouslyas a mediator of insulin resistance in diabetes mellitus [Hotamisligiland Spiegelman, Diabetes 43:1271-1278 (1994)]. In addition, anover-abundance of the active form of TNF-alpha has been linked toadverse symptoms in a number of disease states, including in rheumatoidarthritis, Crohn's disease, sepsis, and cachexia. Since there arepresently no effective treatments for these conditions, there remains aneed to find new drugs that can be used to modulate the effectivephysiological concentration of the active form of TNF-alpha.

TNF-alpha is transcribed as a transmembrane protein having a monomericmolecular weight of 26 kilodaltons [Shirai et al., Nature 313:803-806(1985)]. A metalloprotease, tumor necrosis factor-alpha convertingenzyme (TACE) cleaves the membrane-associated form of TNF-alpha at aspecific site of the protein, converting it to its corresponding solubleform [Black et al., Nature 385:729-733 (1997); Moss et al., Nature385:733-736 (1997); U.S. Pat. No. 5,830,742, Issued Nov. 3, 1998; U.S.Pat. No. 6,013,466, Issued Jan. 11, 2000, the contents of which arehereby incorporated by reference in their entireties]. The soluble formof TNF-alpha then associates as a homotrimer of three 17 kilodaltonmonomers [Kriegler et al., Cell, 53:45-53 (1988)]. Although themembrane-associated form of TNF-alpha appears to be active, it is theproteolyzed soluble form that is responsible for the mortalityassociated with endotoxic shock [Gearing et al., Nature 370:555-558(1994)]. Thus, reducing the circulating concentration of activeTNF-alpha appears to be critical to alleviate the harmful side effectscaused by this cytokine. One means for achieving this reduction inconcentration of soluble TNF-alpha is to inhibit the TACE protease.

TACE, also referred to as ADAM 17 and CD156q, is a zinc endopeptidasethat is a member of the “A Disintegrin And Metalloprotease” (ADAM)family of metalloproteases [Schlondorff and Blobel, J. Cell Sci.,112:3603-3617 (1999); Black, Intern. J. Biochem. Cell Biol 34:1-5(2002); U.S. Pat. No. 5,830,742, Issued Nov. 3, 1998, the contents ofall of which are hereby incorporated by reference in their entireties].A type I transmembrane protein, TACE comprises (i) an extracellularregion having an N-terminal signal peptide, (ii) a pro domain, (iii) azinc-dependent catalytic domain, (iv) a disintegrin domain, (v) anEGF-like domain, (vi) a crambin-like domain, (vii) a transmembrane helixand (viii) an intracellular C-terminal tail [see WO9940182, PublishedAug. 12, 1999]. More recently, the EGF-like and the crambin-like domainshave been grouped together and re-named as a cysteine-rich domain[Black, Intern. J. Biochem. Cell Biol 34:1-5 (2002)].

Since TACE is a protease, the portion of the enzyme that is critical fordrug discovery is its catalytic domain. The catalytic domain (TCD) ofTACE comprises 263 amino acid residues preceded by a furin cleavage site(residues 211-214). The pro domain comprises a cysteine that interactswith the zinc molecule at the active-site preventing proteolytic action.Therefore, this cysteine must be displaced in order to generate anactive protease [Black, Intern. J. Biochem. Cell Biol 34:1-5 (2002)].

Zask et al. have prepared a compilation of inhibitors ofmetalloproteinases [Curr. Pharm. Des., 2:624-661 (1996), the contents ofwhich are hereby incorporated by reference in their entireties], andmore recently, Letavic et al. has disclosed several specific inhibitorsof TACE [Biorgan. & Medic. Chem Lett. 12:1387-1390 (2002), the contentsof which are hereby incorporated by reference in their entireties]. Todate, however, none of these has proven to be useful in the treatment ofconditions related to an over-abundance of soluble TNF-alpha.

Three-dimensional structures of two different TACE-inhibitor complexeshave been obtained via X-ray crystallographic analyses [Letavic et al.,Biorgan. & Medic. Chem Lett. 12:1387-1390 (2002); WO9940182, PublishedAug. 12, 1999, U.S. application Ser. No. 09/117,476, filed Jan. 27,1999, the contents of which are all hereby incorporated by reference intheir entireties]. Importantly, however, the conditions for preparingthe two TACE-inhibitor complexes were significantly different. Theseresults suggest that new crystallization conditions may be required forevery different TACE-ligand complex. Determining crystallizationconditions, de novo can be extremely time-consuming. Moreover, such arequirement severely hampers progress in identifying new and more potentinhibitors of TACE which are necessary for developing safe and effectivedrugs to ameliorate the deleterious effects due to an overabundance ofthe soluble form of TNF-alpha.

Therefore, there is need to provide methods for performing X-raycrystallographic structural determinations on multiple TACEprotein-ligand complexes without having to determine crystallizationconditions, de novo. In addition, there is a need to obtain X-raydiffractable crystals of the TACE catalytic domain that are stable.Furthermore, there is a need to obtain crystals of the TACE catalyticdomain that are amenable to ligand exchange.

The citation of any reference herein should not be construed as anadmission that such reference is available as “prior art” to the instantapplication.

SUMMARY OF THE INVENTION

The present invention discloses that the native TACE proteinunexpectedly undergoes autoproteolysis at the high proteinconcentrations required for X-ray crystallography. The present inventionfurther identifies the peptide bond between amino acid residues Tyr352and Val353 (Y₃₅₂–V₃₅₃) of SEQ ID NO: 2 as the specific cleavage site.

Therefore, the present invention provides a polypeptide comprising amodified TACE catalytic domain that is significantly less susceptible toautoproteolysis than the native TACE. The modified TACE of the presentinvention imparts improved stability to the protease under theconditions employed to generate TACE crystals. The present inventionalso uniquely enables X-ray crystallographic structural determinationsto be performed on multiple TACE protein-ligand complexes in rapidsuccession. This ability to rapidly generate three-dimensionalstructures of TACE protein-ligand complexes is critical for a successfulstructure based rational drug design program.

Indeed, the structural information generated using the compositions andmethods of the present invention greatly facilitates the identificationof new and more potent inhibitors of the TACE protease. Selectedinhibitors, in turn, become lead candidates in the development of drugsthat will be useful for counteracting the harmful effects due to anoverabundance of the soluble form of TNF-alpha, i.e., drugs that can beused in the treatment of rheumatoid arthritis, Crohn's disease, sepsis,and/or cachexia.

One aspect of the present invention provides a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 8. In one such embodiment, thepolypeptide consists essentially of the amino acid sequence of SEQ IDNO: 8. In a particular embodiment, the amino acid residue at position139 of SEQ ID NO: 8 is serine.¹ In another embodiment of this type, theamino acid residue at position 139 of SEQ ID NO: 8 is alanine. In apreferred embodiment, the amino acid residue at position 139 of SEQ IDNO: 8 is glycine (denoted as “vgTACE” in the Example below, and has theamino acid sequence of SEQ ID NO: 20).

¹The amino acid sequences of the TACE catalytic domain and the modifiedTACE catalytic domain are SEQ ID NOs: 6 and 8, respectively. Position139 of SEQ ID NOs: 6 and 8 is equivalent to position 353 of SEQ ID NO:2, the amino acid sequence of the TACE polypeptide comprising the Pre,Pro and catalytic domains.

In a related embodiment, the present invention provides a polypeptidecomprising a modified TACE catalytic domain comprising at least 95%identity with the amino acid sequence of SEQ ID NO: 8. Preferably, thispolypeptide catalyzes the proteolytic cleavage of SEQ ID NO: 17 and/orbinds toN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide and/or binds toN-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH. In onesuch embodiment, the polypeptide consists essentially of a modified TACEcatalytic domain having at least 95% identity with the amino acidsequence of SEQ ID NO: 8. In one specific embodiment of this type, theamino acid residue at position 139 of SEQ ID NO: 8 is alanine. Inanother embodiment, the amino acid residue at position 139 of SEQ ID NO:8 is glycine. In yet another embodiment, the amino acid residue atposition 139 of SEQ ID NO: 8 is serine.

The present invention also provides the full-length TACE polypeptide andfragments thereof that comprise the TACE modified catalytic domain. Inone such embodiment the TACE protein comprises (i) the pre domain, (ii)the pro domain, and (iii) the modified catalytic domain. In anotherembodiment, the fragment of the TACE polypeptide comprises the prodomain, and the modified catalytic domain.

Chimeric proteins comprising the polypeptides of the present inventionare also part of the present invention. In a particular embodiment, thechimeric TACE is a fusion protein comprising (i) the pre domain, (ii)the pro domain, (iii) the modified catalytic domain and (iv) apolyhistidine tag. In another embodiment, the chimeric protein comprisesthe modified catalytic domain and a polyhistidine tag. In a preferredembodiment, the polyhistidine tag further comprises a glycyl-seryl(i.e., Gly-Ser) linker.

The present invention further provides nucleic acids that encode thepolypeptides and chimeric proteins of the present invention (see e.g.,Table 1 below). In a particular embodiment, the nucleic acid encodes apolypeptide having the amino acid of SEQ ID NO: 8. The present inventionfurther provides expression vectors that comprise the nucleic acids ofthe present invention and a transcriptional control sequence. Preferablythe nucleic acids of the present invention are operatively linked to thetranscriptional control sequences in the expression vectors. Host cellscomprising the expression vectors are also part of the presentinvention.

In addition, the present invention provides methods for producing theabove-mentioned polypeptides. One such embodiment comprises culturing ahost cell of the present invention that produces the polypeptides.Methods for purifying the resulting recombinant polypeptides are alsoincluded in the present invention, as are the purified recombinantpolypeptides.

Crystals, each comprising one of the protein-ligand complexes of thepresent invention, are also contemplated. Preferably, such crystalseffectively diffract X-rays for the determination of the atomiccoordinates of the protein-ligand complex to a resolution of greaterthan 5.0 Angstroms. More preferably, a crystal of the present inventioneffectively diffracts X-rays for the determination of the atomiccoordinates to a resolution of greater than 3.5 Angstroms. Even morepreferably, a crystal of the present invention effectively diffractsX-rays for the determination of the atomic coordinates equal to orgreater than 3.0 Angstroms. Still more preferably, a crystal of thepresent invention effectively diffracts X-rays for the determination ofthe atomic coordinates equal to or greater than 2.5 Angstroms, and, yeteven more preferably, equal to or greater than 2.0 Angstroms. Mostpreferably, a crystal of the present invention effectively diffractsX-rays for the determination of the atomic coordinates equal to orgreater than 1.5-1.7 Angstroms.

In a particular embodiment, the crystal comprises a protein-ligandbinding complex in which the polypeptide comprises the amino acidsequence of SEQ ID NO: 8. In another embodiment, the crystal comprises apolypeptide comprising a protein-ligand binding complex in which thepolypeptide comprises a modified TACE catalytic domain having at least95% identity with the amino acid sequence of SEQ ID NO: 8. Preferablythis polypeptide catalyzes the proteolytic cleavage of SEQ ID NO: 17,and/or binds to the compound,N-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide and/or binds toN-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH. In oneparticular embodiment of this type, the crystal of the present inventionhas the space group P2₁2₁2₁, with unit cell dimensions of: a=73, b=75,c=103 Angstroms.

Another aspect of the present invention provides methods for obtaining acrystal comprising a protein-ligand complex between a substitute ligandand a modified TACE catalytic domain. One such method comprisesincubating (e.g., soaking) an excess of a substitute ligand with acrystal comprising a modified TACE catalytic domain and an initialligand in a protein-ligand binding complex. The incubation is performedunder the appropriate conditions and for a sufficient time period forthe substitute ligand to replace the initial ligand in theprotein-ligand complex. A crystal comprising the protein-ligand complexbetween the substitute ligand and the modified TACE catalytic domain isthus, obtained. The modified TACE catalytic domain can be part of alarger polypeptide (e.g., the full-length TACE or a chimeric protein).In a preferred embodiment, the modified TACE catalytic domain comprisesthe amino acid sequence of SEQ ID NO: 8. In a one such embodiment, themodified TACE catalytic domain comprises the amino acid sequence of SEQID NO: 20. In a preferred embodiment, the initial ligand isN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide. In a particular embodiment of this type, thesubstitute ligand isN-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH. Thepresent invention further provides the crystals that compriseprotein-ligand binding complexes that have had their initial ligandreplaced with a substitute ligand.

In an alternative embodiment, the present invention provides a methodfor identifying an agent for use as an inhibitor of TACE. One suchembodiment comprises obtaining a set of atomic coordinates that definethe three-dimensional structure of the protein-ligand binding complexfrom a crystal of the present invention. A potential agent is thenselected by performing rational drug design with the atomic coordinatesobtained. Preferably, the selection is performed in conjunction withcomputer modeling. The potential agent is contacted with a proteolyticpolypeptide that comprises the catalytic domain of TACE, oralternatively, an active fragment thereof. The catalytic activity of theproteolytic polypeptide is then determined in a TACE activity assay. Apotential agent is identified as an agent that inhibits TACE when thereis a decrease in the activity of the proteolytic polypeptide in thepresence of the agent relative to in its absence.

In a related embodiment, a method of identifying a compound that ispredicted to inhibit TACE is provided. One such embodiment comprisesusing the atomic coordinates in Table 3 to define the structure of thecomplex between the modified catalytic domain of TACE andN-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH and/orto define the structure of a portion of that complex. The portion of thecomplex defined is required to comprise sufficient structuralinformation to enable the identification of a given compound as apotential inhibitor. A compound then is identified as one predicted toinhibit TACE through the use of the defined structure. A particularembodiment of this type further comprises contacting the compound with aproteolytic polypeptide comprising the catalytic domain of TACE or anactive fragment thereof and determining the catalytic activity of theproteolytic polypeptide in a TACE activity assay. A potential agent isidentified as an agent that inhibits TACE when there is a decrease inthe activity of the proteolytic polypeptide in the presence of the agentrelative to in its absence.

The present invention further provides a computer comprising athree-dimensional representation of a protein-ligand complex between amodified TACE catalytic domain andN-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH incomputer memory. One such embodiment comprises: (i) a machine-readabledata storage medium comprising a data storage material encoded withmachine-readable data comprising the atomic coordinates of Table 3; (ii)a working memory for storing instructions for processing themachine-readable data; and (iii) a central-processing unit coupled tothe working memory and to the machine-readable data storage medium forprocessing the machine readable data into a three-dimensionalrepresentation. Preferably a display is also provided that is coupled tothe central-processing unit for visualizing the three-dimensionalrepresentation of the protein-ligand complex.

Accordingly, it is a principal object of the present invention toprovide an active TACE catalytic domain that can form a stable X-raydiffractable crystal.

It is a further object of the present invention to provide a way forexchanging ligands of a TACE catalytic domain in a crystal.

It is a further object of the present invention to provide multiplecrystals of the TACE catalytic domain each comprising a differentprotein-ligand complex.

It is a further object of the present invention to provide an effectiveway of performing structure based rational drug design with TACE.

It is a further object of the present invention to provide drugs totreat conditions due to an overabundance of the soluble form ofTNF-alpha.

These and other aspects of the present invention will be betterappreciated by reference to the following drawing and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts a schematic of a computer comprising a central processingunit (CPU), a working memory, a mass storage memory, a display terminal,and a keyboard that are interconnected by a conventional bidirectionalsystem bus. The computer can be used to display and manipulate thestructural data of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Although entirely independent of any particular mechanism, the presentinvention was conceived following the unexpected discovery by thepresent inventors that the native TACE protein undergoes autoproteolysisat the high protein concentrations required for X-ray crystallographicanalysis. The subsequent identification by the inventors of anautoproteolysis site between Y₃₅₂–V₃₅₃ of SEQ ID NO: 2, raised thepossibility that the replacement of either one or both of these aminoacid residues might lead to a TACE protein that was resistant toautoproteolysis. Insertion(s) or deletion(s) of amino acid residuesadjacent to the Y₃₅₂–V₃₅₃ cleavage site might also lead to a TACEpolypeptide that is resistant to autoproteolyisis. However, inserting ordeleting amino acid residues adjacent to the Y₃₅₂–V₃₅₃ cleavage sitecould create conformational changes in the protein that significantlyreduce its enzyme activity and/or stability. Similarly, since Tyr352 ofSEQ ID NO: 2 is located within a hydrophobic pocket that is close to theactive site of the TACE protease, modification of this amino acidresidue also might effect the enzymatic activity and/or proteinstability. In direct contrast, Val353 of SEQ ID NO: 2 is located on theenzyme surface with its side chain exposed to the solvent, and somodification of the valine side chain might be less traumatic to theenzyme structure. Therefore, modification of Val353 is preferred overeither modifying Tyr352, or inserting or deleting amino acid residuesadjacent to the Y₃₅₂–V₃₅₃ cleavage site. These latter two alternatives,however, remain part of the present invention.

Indeed, as disclosed herein, substitution of the hydrophobic valine sidechain with either serine or glycine significantly reducesautoproteolysis, and dramatically improved the stability of the protein,without significantly altering the proteolytic activity of the TACEenzyme. In a preferred embodiment, the modified TACE catalytic domaincontains an amino acid change at amino acid residue 353 of SEQ ID NO: 2.In one such embodiment, Val353 is replaced with a glycyl residue. Inanother embodiment, Val353 is replaced with a seryl residue.Substitutions of the nonpolar side chain of valine with alternativenon-hydrophobic side chains can also prevent auto-proteolysis, and suchsubstitutions are also part of the present invention. In addition, inorder to remove N-glycosylation sites it is preferable as exemplifiedbelow, that Ser266 be replaced e.g., with an alanyl residue, and Asp452be replaced e.g., with a glutaminyl residue.

The present invention further provides crystals comprising aprotein-ligand complex of a polypeptide that comprises a modified TACEcatalytic domain. The three-dimensional structure of a protein-ligandcomplex comprising a modified TACE catalytic domain bound toN-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH isprovided in the Example below (see Table 3 which lists the atomiccoordinates).

N-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH which iscommercially available, e.g., from Chem-Impex International, Wood DaleIll., (product code 09538), has the following chemical structure:

Structure based rational drug design is the most efficient method ofdrug development. In one common paradigm, a three dimensional structureof a protein-ligand complex is determined, and potential antagonists(e.g., inhibitors) of the protein (e.g., potential drugs) are identifiedand/or designed with the aid of computer modeling [Bugg et al.,Scientific American, Dec.: 92-98 (1993); West et al., TIPS, 16:67-74(1995); Dunbrack et al., Folding & Design, 2:27-42 (1997)]. The drugcandidates are selected and assayed. The most promising drug candidatesare identified, and then incubated in excess with crystals of aprotein-ligand complex to replace the initial ligand. Thethree-dimensional structure of the new protein-ligand complex is thendetermined, and new potential antagonists of the protein are identifiedand/or designed with the aid of computer modeling. This process can thenbe continued in successive iterations until a lead drug candidate isidentified.

Heretofore, the ability to perform structure based rational drug designwith TACE was severely hampered because only two TACE protein-ligandscomplexes were known to form an X-ray quality crystal, [Maskos et. al.,Proc. Natl. Acad. Sci. USA 95:3408-3412 (1998); Letavic et al., Biorgan.& Medic. Chem Lett. 12:1387-1390 (2002)], and these crystals were notreported to be capable of ligand exchange. As disclosed herein, thepresent invention overcomes this problem by providing crystals of themodified TACE catalytic domain that are conducive to ligand exchange.

As used herein the following terms shall have the definitions set outbelow:

As used herein the term “polypeptide” is used interchangeably with theterm “protein” and is further meant to encompass peptides. Therefore, asused herein, a polypeptide is a polymer of two or more amino acidsjoined together by peptide linkages. Preferably, a polypeptide is apolymer comprising twenty or more amino acid residues joined together bypeptide linkages.

As used herein a polypeptide “consisting essentially of” or that“consists essentially of” a specified amino acid sequence is apolypeptide that (i) retains the general characteristics of apolypeptide comprising that amino acid sequence, e.g., the activity ofthe polypeptide, and (ii) further comprises the identical amino acidsequence, except it consists of plus or minus 10% (or a lowerpercentage), preferably plus or minus 5% (or a lower percentage), andmore preferably plus or minus 2.5% (or a lower percentage) of the aminoacid residues.

As used herein a “modified TACE catalytic domain” is a TACE catalyticdomain that has been modified to resist and/or prevent autocatalysis.Preferably, at least one of the two critical amino acid residues at theautoproteolytic site of TACE, i.e., Tyr352 and Val353 of SEQ ID NO: 2,has been replaced. More preferably, a modified TACE catalytic domain hasthe Val353 residue replaced with a non-hydrophobic amino acid residue.

As used herein a “non-hydrophobic amino acid” is an amino acid that isnot hydrophobic. The genus of non-hydrophobic amino acids specificallydoes not include leucine, isoleucine, valine, methionine, tryptophan,and phenylalanine.

As used herein a “polypeptide comprising a modified TACE catalyticdomain”, can be (i) the full length TACE protein, (ii) a fragment of theTACE protein that includes the modified TACE catalytic domain e.g., thepro and catalytic domain, (iii) the modified TACE catalytic domainalone, or (iv) a chimeric protein which comprises any of the above.

As used herein a “proteolytic polypeptide” of the present invention is apolypeptide that is capable of catalyzing the proteolytic cleavage of asubstrate of the native TACE protease. A proteolytic polypeptide of thepresent invention minimally comprises an active fragment of the TACEcatalytic domain that retains proteolytic activity. A proteolyticpolypeptide of the present invention can be a chimeric protein.

As used herein an “active fragment” of the catalytic domain of TACE” isa fragment of the catalytic domain of TACE and/or modified TACEcatalytic domain that retains at least about 10%, preferably at leastabout 20%, and more preferably at least about 25% of the proteolyticactivity of the full-length TACE protease. Preferably, the activefragment retains at least about 25%, more preferably at least about 50%,and even more preferably at least about 75% of the amino acid residuesof the catalytic domain of TACE having the amino acid sequence of SEQ IDNO: 6. More preferably, the amino acid sequence of the active fragmentof the TACE catalytic domain has at least about 95% identity to thecorresponding amino acid residues of SEQ ID NO: 6.

As used herein the term “chimeric” protein is meant to include “fusionproteins”. “Chimeric” proteins of the present invention comprise atleast a portion of a non-TACE protein joined via a peptide bond to atleast a portion of a TACE polypeptide. Chimeric proteins can haveadditional structural, regulatory, or catalytic properties. In aparticular embodiment the chimeric protein functions as a means ofdetecting and/or isolating the TACE polypeptide or fragment thereofafter the recombinant nucleic acid is expressed. Non-TACE amino acidsequences are preferably either amino- or carboxy-terminal to the TACEsequence.

As used herein one amino acid sequence is 100% “identical” to a secondamino acid sequence when the amino acid residues of both sequences areidentical. Accordingly, an amino acid sequence is 50% “identical” to asecond amino acid sequence when 50% of the amino acid residues of thetwo amino acid sequences are identical. The sequence comparison isperformed over a contiguous block of amino acid residues comprised bythe TACE polypeptide or the portion of the TACE polypeptide beingcompared, e.g., the modified catalytic domain (SEQ ID NO: 8). In apreferred embodiment, selected deletions or insertions that couldotherwise alter the correspondence between the two amino acid sequencesare taken into account.

As used herein, DNA and protein sequence percent identity can bedetermined using C, MacVector 6.0.1, Oxford Molecular Group PLC (1996)and the Clustal W algorithm with the alignment default parameters, anddefault parameters for identity. These commercially available programscan also be used to determine sequence similarity using the same oranalogous default parameters. Alternatively, an Advanced Blast searchunder the default filter conditions can be used, e.g., using the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program using the default parameters.

As used herein a “nucleic acid” refers to the phosphate ester polymericform of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. When referring to anucleic acid that is double stranded both the “sense” strand and thecomplementary “antisense” strand are intended to be included. Thus anucleic acid that is hybridizable to SEQ ID NO: 1, for example, can beeither hybridizable to the “sense” strand of SEQ ID NO: 1, which isparticularly listed in the SEQUENCE LISTING, or to the “antisense”strand which can be readily determined from that SEQUENCE LISTING.

A DNA “coding sequence” is a double-stranded DNA sequence that istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease SI), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which can then be trans-RNAspliced and translated into the protein encoded by the coding sequence.

A nucleic acid sequence is “operatively linked” to an expression controlsequence when the expression control sequence controls or regulates thetranscription and translation of that nucleic acid sequence. The termoperatively linked includes having an appropriate start signal.

A “heterologous nucleotide sequence” as used herein is a nucleotidesequence that is added to a nucleotide sequence of the present inventionby recombinant methods to form a nucleic acid that is not naturallyformed in nature. Such nucleic acids can encode chimeric proteins.Alternatively, a heterologous nucleotide sequence can contain a nucleicacid regulatory sequence. Thus a heterologous nucleotide sequence cancomprise non-coding sequences including restriction sites, regulatorysites, promoters and the like. In still another embodiment theheterologous nucleotide can function as a means of detecting anucleotide sequence of the present invention. The present inventionprovides heterologous nucleotide sequences that when combined withnucleotide sequences encoding the TACE proteins, and fragments thereof,are necessary and sufficient to encode all of the chimeric proteins ofthe present invention.

As used herein the phrases “structure based rational drug design”,“structure based drug design”, “structure assisted drug design” and“rational drug design” are used interchangeably. These phrases are meantto convey a particular method of identifying and/or designing a ligand(preferably an inhibitor) for a specific target protein that includesthe use of the three-dimensional structure of that protein and/or itscorresponding protein-ligand complex.

The phrase “binding to” in regard to a ligand binding to a polypeptideis used herein to include any or all such specific interactions thatlead to a protein-ligand binding complex. This can include processessuch as covalent, ionic, hydrophobic and hydrogen bonding, but does notinclude non-specific associations such solvent preferences.

As used herein a “ligand” of a polypeptide is a compound that binds tothe polypeptide in a protein-ligand binding complex. In a specificembodiment of the present invention the polypeptide has an enzymaticactivity and the ligand inhibits that activity when bound to thepolypeptide in a protein-ligand binding complex. Such a ligand is alsotermed an “inhibitor”.

As used herein the term “initial ligand” denotes a ligand in aprotein-ligand complex that is, or can be displaced by a “substituteligand”.

As used herein, a “protein-ligand binding complex” is a specificassociation between a polypeptide and the compound that binds to it. Ina preferred embodiment of the present invention, the ligand is aninhibitor of the polypeptide. In a particular embodiment of this type,the binding of the inhibitor to the polypeptide occurs at the activesite of the polypeptide.

As used herein “incubating a ligand with a crystal” is usedinterchangeably with “soaking a crystal with a ligand”. Incubating aligand with a crystal is the contacting of a ligand with a crystal of apolypeptide under the appropriate conditions and for a sufficient timeperiod (e.g., hours to several days) for the ligand to bind to thecrystalline polypeptide and form a crystalline protein-ligand complex.Such incubating can further and/or alternatively, include contacting anexcess of a substitute ligand with a crystal of a protein-ligand complexunder the appropriate conditions and for a sufficient time period (e.g.,hours to several days) for the substitute ligand to replace the initialligand and form the new crystalline protein-ligand complex.

As used herein the terms “displacing”, “replacing”, and “exchanging” areused interchangeably in regard to the substitution of one ligand in aprotein-ligand complex for another.

As used herein an “excess of a substitute ligand” is an amount of thatligand that is sufficient to replace 80% or more, and preferably 90% ormore, of the initial ligand in a protein-ligand complex. In a particularembodiment of this type, the concentration of the substitute ligand isabout ten-fold higher than the concentration of the protein-ligandcomplex. In a preferred embodiment, the concentration of the substituteligand is about one hundred-fold higher than the concentration of theprotein-ligand complex.

As used herein the term “X-ray diffractable crystal” is a crystal of acompound that yields a discernable diffraction pattern when subjected to0.5 to 2.5 Å incident X-ray radiation.

As used herein an “X-ray quality crystal” is an X-ray diffractablecrystal that can yield meaningful structural data of its crystallinecomposition when subjected to X-ray crystallographic analysis.

As used herein, and unless otherwise specified, the terms “agent”,“potential drug”, “compound”, or “test compound” are usedinterchangeably, and refer to chemicals that have or potentially have ause as an inhibitor of the proteolytic activity of TACE. Preferably suchagents include drugs for the treatment or prevention of a disease and/orcondition involving the proteolytic action of TACE. Therefore, suchagents may be used, as described herein, in drug assays and drug screensand the like.

As used herein a “small organic molecule” is an organic compound [ororganic compound complexed with an inorganic compound (e.g., metal)]that has a molecular weight of less than 3 Kd.

As used herein the terms “approximately” and “about” are used to signifythat a value is within twenty percent of the indicated value i.e., anamino acid sequence containing “approximately” 260 amino acid residuescan contain between 208 and 312 amino acid residues.

Nucleic Acids Encoding TACE

Obtaining and/or constructing a cDNA that encodes a polypeptidecomprising a modified TACE facilitates the production of the largequantities of protein required to perform X-ray crystallographicanalysis. Since the sequence of the native protein is known [see U.S.Pat. No. 6,013,466, Issued Jan. 11, 2000, the contents of which arehereby incorporated by reference in their entireties], a cDNA encodingthe modified protease can be readily obtained.

To express a recombinant protein of the present invention in a hostcell, an expression vector can be constructed comprising thecorresponding cDNA. The present invention therefore, provides expressionvectors containing nucleic acids encoding polypeptides comprising themodified TACE catalytic domains of the present invention. Due to thedegeneracy of nucleotide coding sequences, other DNA sequences whichencode substantially the same amino acid sequence as a nucleic acidencoding a polypeptide comprising the modified TACE catalytic domain ofthe present invention may be used in the practice of the presentinvention. These include, but are not limited to, allelic genes,homologous genes from other species, which are altered by thesubstitution of different codons that encode the same amino acid residuewithin the sequence, thus producing a silent change. Host cellscomprising the expression vectors of the present invention are alsoprovided.

Cloning of cDNAs and expression of their corresponding recombinantproteins have become a routine laboratory exercise [see Sambrook andRussell, Molecular Cloning, A laboratory Manual, 3^(rd) edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor L.I. (2000), thecontents of which are hereby incorporated by reference in theirentireties]. The use of a Baculovirus recombination system and Sf9 hostcells is exemplified below. Purification of recombinant proteins hasalso become a routine laboratory exercise. In the present case, themodified TACE protein was cloned and expressed as the pro-protein. Thepre and pro domains were cleaved during protein expression and secretionby the cells. The catalytic domain was then purified (see Examplebelow).

The nucleotide sequence for open reading frame of TACE with a GS linker,a polyHis tag (H6), and stop codon is shown below. The nucleic acidsequence encoding: (i) the pre domain is underlined, (ii) the pro domainis under-dashed, (iii) the catalytic domain is unmarked and (iv) the GSlinker and the polyHis tag (H6) are double underlined. The stop codon isunderlined with a wavy line.

Any technique for mutagenesis known in the art can be used to convertthe native TACE catalytic domain to a modified domain, including but notlimited to, ill vitro site-directed mutagenesis [Hutchinson et al., J.Biol. Chem., 253:6551 (1978); Zoller and Smith, DNA, 3:479-488 (1984);Oliphant et al., Gene, 44:177 (1986); Hutchinson et al., Proc. Natl.Acad. Sci. U.S.A., 83:710 (1986)]. The use of TAB@ linkers (Pharmacia),etc. and PCR techniques also can be employed for site directedmutagenesis [see Higuchi, “Using PCR to Engineer DNA”, in PCRTechnology: Principles and Applications for DNA Amplification, H.Erlich, ed., Stockton Press, Chapter 6, pp. 61-70 (1989)]. Preferablymutagenesis (i.e., modification) of the TACE catalytic domain isperformed in a two step process [Wang, and Malcolm, BioTechniques26:680-682 (1999)]. In the Example below, two extension reactions wereperformed in separate tubes in the first stage: (i) one containing theforward primer, and (ii) the other containing the reverse primer. Aftertwo cycles, the two reactions are mixed and the standard QuickChangemutagenisis procedure is carried out for an additional 18 cycles.Following amplification, the parental strand is digested with 1Unit ofDpn1 for 1 hour and an aliquot is transformed into DH5-alpha cells[GeneWiz, New York, N.Y.]

The TACE Polypeptide

The amino acid sequence for the TACE polypeptide is shown below. (i) Thepre domain is underlined, (ii) the pro domain is under-dashed, and (iii)the catalytic domain is unmarked. The GS-linker and polyhistidine tag(H6) are not included. The valine residue that is replaced with anon-hydrophobic amino acid residue in the modified TACE polypeptide isin bold. In addition, serine-266 has been replaced by an alanine, andasparagine-452 has been replaced by a glutamine in order to remove theN-linked glycosylation sites.

The amino acid sequence for the catalytic domain of the modified TACEpolypeptide is shown below. Whereas, the native protein comprises VAL₃₅₃(VAL₁₃₉ of SEQ ID NO: 6), this amino acid residue is replaced by anon-hydrophobic amino acid residue in a modified TACE catalytic domain.This amino acid position is denoted with an “X” in bold below.Preferably the non-hydrophobic amino acid residue is a glycl, alanyl, orseryl amino acid residue. The GS-linker and Polyhistidine tag (H6) arenot included.RADPDPMKNTCKLLVVADHRFYRYMGRGEESTTTNYLIELIDRVDDIYRNTAWDNAGFKGYGIQIEQIRILKSPQEVKPGEKHYNMAKSYPNEEKDAWDVKMLLEQFSFDIAEEASKVCLAHLFTYQDFDMGTLGLAYXGSPRANSHGGVCPKAYYSPVGKKNIYLNSGLTSTKNYGKTILTKEADLVTTHELGHNFGAEHDPDGLAECAPNEDQGGKYVMYPIAVSGDHENNKMFSQCSKQSIYKTIESKAQECFQERSNKV (SEQ ID NO: 8), where X is a non-hydrophobicamino acid residue, and preferably either alanine, glycine or serine.

In a particular embodiment of the present invention, a modified TACEpolypeptide or fragment thereof (e.g., the catalytic domain) is at leastabout 75% identical, more preferably at least about 90% identical, andmost preferably at least about 95% identical to the TACE polypeptide orfragment thereof. As indicated above, a modified TACE or fragmentthereof has a non-hydrophobic amino acid residue in place of the valineat position 353 (as defined in SEQ ID NO: 2).

Polypeptides comprising the modified TACE catalytic domains of theinvention include those containing altered sequences in whichfunctionally equivalent amino acid residues are substituted for residueswithin the sequence resulting in a conservative amino acid substitution.For example, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity, which acts as afunctional equivalent, resulting in a silent alteration. Substitutes foran amino acid within the sequence may be selected from other members ofthe class to which the amino acid belongs.

For example, the nonpolar amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.Amino acids containing aromatic ring structures are phenylalanine,tryptophan, and tyrosine. The polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Thepositively charged (basic) amino acids include arginine and lysine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

Particularly preferred conserved amino acid exchanges are:

(a) Lys for Arg or vice versa such that a positive charge may bemaintained;

(b) Glu for Asp or vice versa such that a negative charge may bemaintained;

(c) Ser for Thr or vice versa such that a free —OH can be maintained;

(d) Gln for Asn or vice versa such that a free NH₂ can be maintained;and

(e) Ile for Leu or for Val or vice versa as roughly equivalenthydrophobic amino acids.

All of the modified TACE catalytic domains of the present invention alsocan be part of a chimeric protein. In a specific embodiment, a chimericTACE protein is expressed in a eukaryotic cell. Such a chimeric proteincan be a fusion protein used to isolate a modified TACE of the presentinvention, through the use of an affinity column that is specific forthe protein fused to the TACE protein. In one such embodiment, thechimeric TACE is expressed in a eukaryotic cell. Examples of such fusionproteins include: a glutathione-S-transferase (GST) fusion protein, amaltose-binding (MBP) protein fusion protein, a FLAG-tagged fusionprotein, or as specifically exemplified below, a poly-histidine-taggedfusion protein.

Expression of a chimeric TACE protein, or fragment thereof, as a fusionprotein can facilitate stable expression, and/or allow for purificationbased on the properties of the fusion partner. Thus the purification ofthe recombinant polypeptides of the present invention can be simplifiedthrough the use of fusion proteins having affinity tags. For example,GST binds glutathione conjugated to a solid support matrix, MBP binds toa maltose matrix, and poly-histidine chelates to a NI-chelation supportmatrix, as specifically exemplified below [see Hochuli et al.,Biotechnolgy 6:1321-1325 (1998)]. The fusion protein can be eluted fromthe specific matrix with appropriate buffers, or by treating with aprotease that is specific for a cleavage site that has been geneticallyengineered in between the TACE protein and its fusion partner.Alternatively, a modified TACE catalytic domain can be combined with amarker protein such as green fluorescent protein [Waldo et al., NatureBiotech. 17:691-695 (1999); U.S. Pat. No. 5,625,048 filed Apr. 29, 1997and WO 97/26333, published Jul. 24, 1997, the contents of which arehereby incorporated by reference herein in their entireties].

Alternatively or in addition, other column chromatography steps (e.g.,gel filtration, ion exchange, affinity chromatography etc.) can be usedto purify the recombinant proteins of the present invention. In manycases, such column chromatography steps employ high performance liquidchromatography or analogous methods in place of the more classicalgravity-based procedures.

As exemplified below, a recombinant modified TACE catalytic domain waspurified with a NiNTA column following routine centrifugation anddiafiltration steps. After the purified protein was collected from theNiNTA column, it was placed on a gel filtration column. The resultingeluate was then concentrated and desalted prior to being combined withan inhibitor to form a protein-ligand complex.

Alternatively, polypeptides comprising the modified TACE catalyticdomains of the present invention can be chemically synthesized [seee.g., Synthetic Peptides: A User's Guide, W.H. Freeman & Co., New York,N.Y., pp. 382, Grant, ed. (1992)].

Enzyme Assays

The catalytic activity of the TACE protease can be determined in anassay using the synthetic peptide Ac-SPLAQA-VRSSSR-NH₂ (SEQ ID NO: 17)as the substrate. This amino acid sequence corresponds to the cleavagesite of TACE on proTNF-alpha, with the sessile bond being between thealanine and the valine. The activity can be measured by incubating 100nM TACE with 100 micromolar substrate in 25 mM HEPES pH 7.3, 5 mM CaCl₂,for 1 hour at room temperature. Product formation can be quantified at214 nm by HPLC using a reverse phase column to separate the substratefrom the products. The ability of a given compound added to the reactionto act as an inhibitor of TACE can then be determined.

Alternatively, TACE activity can be determined in a fluorescence assayusing the synthetic peptide substrate, K(Mca)-SPLAQA-VRSSSRK(Dpn)-NH₂(SEQ ID NO: 18). K(Mca) is a lysyl residue modified by comprising anepsilonN-methoxycoumarin, whereas K(Dpn)-NH₂ is a lysyl residue modifiedto comprise an epsilonN 2,4, dinitrophenyl. 2-100 nanomolar TACEprotease (or active fragment thereof) is incubated with 25 micromolarpeptide substrate in 25 mM HEPES pH 7.3, 5 mM CaCl₂ for 1 hour at roomtemperature. Product formation is detected by exciting at 340 nm andmeasuring the fluorescence emission at 380 nm every 30 seconds for aboutan hour. The initial velocity can be obtained by linear regression. Theincrease in fluorescence emission can be correlated with the quantity ofcleaved product. The ability of a given compound added to the reactionto act as an inhibitor of TACE can then be determined.

Crystallization

Crystals of the protein-ligand complex comprising a modified TACEcatalytic domain of the present invention can be grown by a number oftechniques including batch crystallization, vapor diffusion (e.g., bysitting drop or hanging drop) and by microdialysis. In the Examplebelow, the modified TACE catalytic domain was complexed withN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide and crystallized by hanging drop vapor diffusion.Seeding of the crystals in some instances is required to obtain X-rayquality crystals. Standard micro and/or macro seeding of crystals maytherefore be used.

As exemplified below, the protein-ligand complex comprising the modifiedTACE catalytic domain V353G (vgTACE) was crystallized under similarconditions to those previously employed for the non-modified TACE[WO9940182, Published Aug. 12, 1999, U.S. application Ser. No.09/117,476, filed Jan. 27, 1999, the contents of which are both herebyincorporated by reference in its entireties].

A substitute ligand can replace the co-crystallized initial ligand bysoaking a crystal of protein-initial ligand complex with the substituteligand. Thus, one or more crystals of protein-initial ligand complex canbe placed in the reservoir solution containing about a 10-fold orgreater excess of substitute ligand. The crystal is kept under theappropriate conditions and for a sufficient time period for thesubstitute ligand to replace the initial ligand and form the newcrystalline protein-substitute ligand complex. In the example below, acrystal was kept in the solution containing the substitute ligand forabout 72 hours. After the incubation, the crystal of theprotein-substitute ligand complex can be frozen in liquid propane, forexample and then used for X-ray diffraction.

Crystals can be characterized using X-rays produced in a conventionalsource (such as a sealed tube or a rotating anode) or using asynchrotron source. Methods of characterization include, but are notlimited to, precision photography, oscillation photography anddiffractometer data collection. As exemplified below, the crystals wereflash frozen in liquid propane and X-ray diffraction was collected at100 degrees Kelvin using conventional or synchrotron sources.

In the Example below, the crystal structure of the modified TACEcatalytic domain V353G (vgTACE) was solved by molecular replacement andthen refined using standard crystallographic programs. The publishedTACE structure was used as the starting model [PDB code:1BKC; Maskos et.al., Proc. Natl. Acad. Sci. USA 95:3408-3412 (1998); WO9940182,Published Aug. 12, 1999, U.S. application Ser. No. 09/117,476, filedJan. 27, 1999, the contents of which are both hereby incorporated byreference in its entirety]. Replacement of the co-crystallized inhibitorwas verified by difference electron density maps. The vgTACE:inhibitorstructures were refined using X-PLOR [Brunger et al., Acta Crystallogr.A 46:585-593 (1990); Brunger et al., Acta Crystallogr. D Biol.Crystallogr., 54:905-921 (1998)].

Refinement calculations also can be performed using CNS [Adams et al.,Proc. Natl. Acad. Sci. USA, 94:5018-5023 (1997)]. Map interpretation andmodel building also can be performed using O [Jones et al., Acta Cryst,A 47:110-119 (1991)]. Other computer programs that can be used to solvecrystal structures include: QUANTA, CHARMM; INSIGHT; SYBYL; MACROMODE;and ICM.

Generally, structure based rational drug design is performed byanalyzing the three-dimensional structures of successive protein-ligandcomplexes. This iterative process requires X-ray quality crystals ofnumerous protein-ligand complexes. These crystals can be obtained threeways. First, crystals of each protein-ligand complex can be grown denovo. This is the most time-consuming method, and in many instancesrequires determining a new set of crystallization conditions. The secondmethod is to incubate (e.g., soak) individual crystals of theuncomplexed protein with each different ligand. This method is muchfaster than growing new crystals, but still requires a relatively largestock of protein to generate all of the new crystals. The third and mostexpedient method is to incubate a previously formed protein-ligandcrystal with a large excess of a substitute ligand, thereby replacingthe initial ligand with the substitute ligand in the protein-ligandcomplex. Heretofore, it was difficult to prepare alternativeprotein-ligand complexes of TACE since the two available X-ray qualitycrystals of TACE comprised the unstable, native TACE. The presentinvention overcomes this problem by providing a modified TACE catalyticdomain that forms X-ray quality crystals that are amenable to ligandexchange.

Structure Based Rational Drug Design

Once three-dimensional structures of crystals comprising modified TACEcatalytic domains are determined, a potential inhibitor of TACE can beexamined through the use of computer modeling using a docking programsuch as GRAM, DOCK, or AUTODOCK [Dunbrack et al., Folding & Design,2:27-42 (1997)]. This procedure can include computer fitting ofpotential inhibitors to the modified TACE catalytic domain to ascertainhow well the shape and the chemical structure of the potential modulatorwill interact with the TACE protein [Bugg et al., Scientific American,Dec.:92-98 (1993); West et al., TIBS, 16:67-74 (1995)]. Computerprograms can also be employed to estimate the attraction, repulsion, andsteric hindrance of the modified TACE catalytic domain with aninhibitor.

Generally the tighter the fit, the lower the steric hindrances, and thegreater the attractive forces, the more potent the inhibitor, sincethese properties are consistent with a tighter binding constant.Furthermore, the more specificity in the design of a potential drug themore likely that the drug will not interact as well with other proteins.This will minimize potential side-effects due to unwanted interactionswith other proteins.

Initially compounds known to bind TACE, for exampleN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide, or a compound that inhibits TACE disclosed byLetavic et al., [Biorgan. & Medic. Chem Lett. 12:1387-1390 (2002) thecontents of which are hereby incorporated by reference in theirentireties], or alternatively, a compound that binds metalloproteases asdisclosed as by Zask et al. [Curr. Pharm. Des., 2:624-661 (1996), thecontents of which are hereby incorporated by reference in theirentireties], can be systematically modified by computer modelingprograms until one or more promising potential analogs are identified.Such analysis has been shown to be effective in the development of HIVprotease inhibitors [Lam et al., Science 263:380-384 (1994); Wlodawer etal., Ann. Rev. Biochem. 62:543-585 (1993); Appelt, Perspectives in DrugDiscovery and Design 1:23-48 (1993); Erickson, Perspectives in DrugDiscovery and Design 1:109-128 (1993)]. Alternatively, a potentialinhibitor initially can be obtained by screening a random peptidelibrary or a chemical library. In the former case, a random peptidelibrary can be produced by recombinant bacteriophage, for example,[Scott and Smith, Science, 249:386-390 (1990); Cwirla et al., Proc.Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al., Science,249:404-406 (1990)]. A peptide selected in this manner would then besystematically modified by computer modeling programs, as describedabove.

If a potential inhibitor is a small organic compound, it can be selectedfrom a library of chemicals, including commercially available chemicallibraries. Alternatively, the small organic compound may be synthesizedde novo. The de novo synthesis of one or even a relatively small groupof specific compounds is reasonable in the art of drug design. Onceobtained, the potential inhibitor can be further tested in a standardbinding and/or catalytic assay with TACE, the TACE catalytic domain, oran active fragment thereof.

For example, a binding assay can be performed following the attachmentof the TACE catalytic domain to a solid support. Methods for placing theTACE catalytic domain on the solid support are well known in the art andinclude such things as linking biotin to the TACE catalytic domain andlinking avidin to the solid support. The solid support can be washed toremove unbound protein. A solution of a labeled potential inhibitor canbe contacted with the solid support. The solid support is washed againto remove the potential inhibitor not bound to the support. The amountof labeled potential inhibitor remaining with the solid support, andthereby bound to the TACE catalytic domain can be determined.Alternatively, or in addition, the dissociation constant between thelabeled potential inhibitor and the TACE catalytic domain, for example,can be determined. Suitable labels for either the TACE catalytic domainor the potential inhibitor include, radioactive labels (e.g., ¹⁴C, ¹H,)and fluorescent labels such as fluorescein isothiocyanate (FITC).

In another embodiment, a Biacore machine can be used to determine thebinding constant of the TACE catalytic domain with a potential inhibitor[O'Shannessy et al. Anal. Biochem. 212:457-468 (1993); Schuster et al.,Nature 365:343-347 (1993)]. In another aspect of the present invention apotential inhibitor is tested for its ability to inhibit the proteolyticactivity of TACE or an active fragment thereof. An inhibitor is thenselected on the basis of its ability to inhibit the catalytic reactionof the TACE protease.

When a promising inhibitor is identified, a crystal comprising aprotein-ligand complex of the inhibitor and the modified TACE catalyticdomain can be prepared by incubating an excess of the inhibitor(substitute ligand) with a crystal of a modified TACE catalyticdomain-ligand complex. The three-dimensional structure of the resultingcrystalline protein-substitute ligand complex can then be determined bymolecular replacement analysis, for example.

Molecular replacement involves using a known three-dimensional structureas a search model to determine the structure of a closely relatedmolecule or protein-ligand complex in a different crystalline form. Themeasured X-ray diffraction properties of the new crystal are comparedwith the search model structure to compute the position and orientationof the protein in the new crystal. Computer programs that can be usedinclude: X-PLOR (see above), CNS, (Crystallography and NMR System, anext level of XPLOR), and AMORE [Navaza, Acta Crystallographics ASO,157-163 (1994)]. Once the position and orientation are known, anelectron density map can be calculated using the search model to provideX-ray phases. Thereafter, the electron density is inspected forstructural differences and the search model is modified to conform tothe new structure. Using this approach, it is possible to solve thethree-dimensional structures of crystals of any protein-ligand complexof the modified TACE catalytic domain.

For all of the drug screening assays described herein, furtherrefinements to the structure of the drug will generally be necessary andcan be made by the successive iterations of any and/or all of the stepsprovided by the particular drug screening assay and/or in combinationwith other such drug screening assays.

A candidate drug selected by performing structure based rational drugdesign can then be assayed in situ and/or in vivo. A candidate drug canbe identified as a drug, for example, if it ameliorates a symptom causedby an overabundance of the soluble form of TNF-alpha in an animal model.Indeed, methods of testing such potential candidate drugs in animalmodels are well known in the art. The potential drugs can beadministered by a variety of ways including topically, orally,subcutaneously, or intraperitoneally depending on the proposed use.Generally, at least two groups of animals are used in the assay, with atleast one group being a control group that is administered theadministration vehicle without the potential drug.

Electronic Representation of the Three Dimensional Structure of TACE

The present invention provides the three-dimensional depiction of theTACE catalytic domain in a complex with an inhibitor on an electronicand/or magnetic medium. More specifically, the present inventionprovides the data comprised in Table 3 on an electronic and/or magneticmedium. In addition, the present invention provides a computer thatcomprises a representation of the TACE catalytic domain-inhibitorcomplex in computer memory that can be used to screen for compounds thatwill inhibit the proteolytic activity of TACE. The computer may compriseportions of, or all of the information contained in Table 3. In aparticular embodiment, the computer comprises: (i) a machine-readabledata storage material encoded with machine-readable data, (ii) a workingmemory for storing instructions for processing the machine readabledata, (iii) a central processing unit coupled to the working memory andthe machine-readable data storage material for processing the machinereadable data into a three-dimensional representation, and (iv) adisplay coupled to the central processing unit for displaying thethree-dimensional representation. Thus the machine-readable data storagemedium comprises a data storage material encoded with machine readabledata which can comprise portions of, or all of the structuralinformation contained in Table 3.

One embodiment for manipulating and displaying the structural dataprovided by the present invention is schematically depicted in FIG. 1.As depicted the System 1, includes a computer 2 comprising a centralprocessing unit (“CPU”) 3, a working memory 4 which may be random-accessmemory or “core” memory, mass storage memory 5 (e.g., one or more diskor CD-ROM drives), a display terminal 6 (e.g., a cathoderay tube), oneor more keyboards 7, one or more input lines 10, and one or more outputlines 20, all of which are interconnected by a conventionalbidirectional system bus 30.

Input hardware 12, coupled to the computer 2 by input lines 10, may beimplemented in a variety of ways. Machine-readable data may be inputtedvia the use of one or more modems 14 connected by a telephone line ordedicated data line 16. Alternatively or additionally, the inputhardware may comprise CD-ROM or disk drives 5. In conjunction with thedisplay terminal 6, the keyboard 7 may also be used as an input device.Output hardware 22, coupled to computer 2 by output lines 20, maysimilarly be implemented by conventional devices. Output hardware 22 mayinclude a display terminal 6 for displaying the three dimensional data.Output hardware might also include a printer 24, so that a hard copyoutput may be produced, or a disk drive or CDROM 5, to store systemoutput for later use, [see also U.S. Pat. No. 5,978,740, Issued Nov. 2,1999, the contents of which are hereby incorporated by reference intheir entireties].

In operation, the CPU 3 (i) coordinates the use of the various input andoutput devices 12 and 22; (ii) coordinates data accesses from massstorage 5 and accesses to and from working memory 4; and (iii)determines the sequence of data processing steps. Any of a number ofprograms may be used to process the machine-readable data of thisinvention.

TABLE 1 TABLE OF SEQUENCES SEQ ID NO: Type Description  1 N.A. Pre, Pro,and Catalytic domain  2 A.A. Pre, Pro, and Catalytic domain  3 N.A. Proand Catalytic domain  4 A.A. Pro, and Catalytic domain  5 N.A. Catalyticdomain  6 A.A. Catalytic domain  7 N.A. Catalytic domain (modified)  8A.A. Catalytic domain (modified)  9 N.A. open reading frame 10 N.A.BamH1f primer 11 N.A. Kpn1r primer 12 N.A. V353Gf primer 13 N.A. V353Grprimer 14 N.A. V353Sf primer 15 N.A. V353Sr primer 16 A.A  internalcleave site 17 A.A  synthetic TACE substrate 18 A.A  synthetic TACEsubstrate 19 N.A. vgTACE 20 A.A  vgTACE

The present invention may be better understood by reference to thefollowing non-limiting Example, which is provided as exemplary of theinvention. The following example is presented in order to more fullyillustrate the preferred embodiments of the invention. It should in noway be construed, however, as limiting the broad scope of the invention.

EXAMPLE Material and Methods

Cloning of TACE and TACE Mutants:

The TACE protein was cloned and expressed as the pro-protein. The preand pro domains are cleaved during protein expression and secretion bythe cells. Only the catalytic domain was purified.

Two PCR primers were used to amplify the pre-pro-cat domains of TACEhaving BamH1 and GS-(His)6-Kpn1 sites at the 5′ and 3′ endsrespectively:

BamH1f: 5′ cgcggatccatgaggcagtctctcctattcctg 3′ SEQ ID NO: 10

Kpn1r: 5′ ccggcctaccttagtgatgqtgatgatggtgggatc 3′ SEQ ID NO: 11

The purified PCR fragment was digested with BamH1 and Kpn1 andsubcoloned into the pFastBac1 vector provided in the Bac-to-BacBaculovirus expression system (Invitrogen, Carlsbad, Calif.). The TACEmutants (V353G, V353S) were generated using the QUICKCHANGE kit(Stratagene, La Jolla, Calif., USA) using the native TACE pFastBac1vector as a template and the following complementary mutagenic primers:

V353Gf: SEQ ID NO: 12

5′ GGA ACT CTT GGA TTA GCT TAT GGA GGC TCT CCC AGA GCA AAC 3′

V353Gr: SEQ ID NO: 13

5′ GTT TGC TCT GGG AGA GCC TCC ATA AGC TAA TCC AAG AGT TCC 3′

V353Sf: SEQ ID NO: 14

5′GGA ACT CTT GGA TTA GCT TAT AGC GGC TCT CCC AGA GCA AAC3′

V353Sr: SEQ ID NO: 15

5′ GTT TGC TCT GGG AGA GCC GCT ATA AGC TAA TCC AAG AGT TCC3′

The mutagenesis was performed in two steps as previously described[Wang, and Malcolm, BioTechniques 26:680-682 (1999) the contents ofwhich are hereby incorporated by reference in their entireties]. In thefirst stage, two extension reactions were performed in separate tubes;one containing the forward primer and the other containing the reverseprimer. After two cycles, the two reactions were mixed and the standardQUICKCHANGE mutagenisis procedure was carried out for an additional 18cycles. Following amplification, the parental strand was digested with1Unit of Dpn1 for 1 hour and an aliquot was transformed into DH5-alphacells. The sequences of all of the vectors were confirmed. (GeneWiz, NewYork, N.Y.)

Production of Recombinant Baculovirus. Recombinant baculovirus wasproduced using the BAC-TO-BAC expression system (Invitrogen, Carlsbad,Calif.) following known protocols for the transposition, isolation andtransfection of recombinant bacmid DNA into Sf9 cells for production ofviral particles. The virus was amplified to the P2 generation and wastitered using the BacPAC Baculovirus Rapid Titer Kit (Clonetech, PaloAlto, Calif.).

Expression and Purification of TACE and TACE mutants: Logarithmicallygrowing Trichoplusia Ni cells (High-5™ cells, 2×10⁶ cells/ml) wereinfected with amplified baculovirus at a MOI=1.0 (2.5×10⁸ pfu/ml) andgrown at 27 degrees Celsius for 48-60 hours. Secreted TACE was isolatedfrom the cell culture media after clarification by centrifugation. Thepooled supernatants were concentrated 10 fold and the buffer exchangedinto 25 mM HEPES, 0.15M NaCl, pH 7.5 by diafiltration. To the desaltedsupernatant, 4-aminophenyl-mercuric acetate (APMA) was added to 20 μM,lauryl maltoside to 0.05%, and imidazole to 25 mM. The supernatant wasthen applied to a NiNTA column (Qiagen Hilden, Germany). The NiNTAcolumn was washed with 25 mM imidazole in buffer A (50 mM HEPES, 10%glycerol, 0.3M NaCl, 0.1% m-octyl-Beta-D-glucopyranoside, pH 7.5) untila stable baseline was achieved. The protein was then eluted with 250 mMimidazole in buffer A. The eluted protein was diluted to 0.1 mg/ml anddialyzed overnight against 25 mM Tris pH 7.5, with 20 μM APMA to digestexcess pro-domain. The protein was collected and adjusted to 0.15M NaCl,concentrated, and applied to a SUPERDEX-75 gel filtration column(Pharmacia) equilibrated with 25 mM Tris-HCl, 0.2M NaCl pH 7.5 at 4degrees Celsius. Fractions corresponding to the monomer of TACE werepooled, and stored at 4 degrees Celsius. The pooled TACE enzyme wasconcentrated to 15 mg/ml, desalted into 25 mM Tris-HCl pH 7.5 usingBIO-SPIN 6 columns (Bio-Rad, Hercules, Calif.) and immediately complexedwithN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide at a 1:1.5 molar ratio. Using this protocol, totalexpression levels of 5-10 mg/L were obtained with a final recovery of0.5–5 mg/L.

Crystallization: Crystals were obtained by the hanging drop vapordiffusion method [Ducruix and Giege. Crystallization of Nucleic Acidsand Proteins. A practical approach. Oxford University Press, (1992)]. Asmall volume (1 to 15 microliters) containing theN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide [commercially available from e.g., CALBIOCHEM, SanDiego Calif., Catalogue No. 579052, (TAPI-2)] solution was equilibratedwith a larger volume (1 ml) of a reservoir solution. The reservoirsolution contains the precipitant that facilitates the crystallization.

During equilibration the water content in the hanging drop is reducedand the protein-ligand complex, [i.e., the complex between vgTACE andN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide] forms crystals. To crystallize the protein-ligandcomplex, one microliter of the protein-ligand complex solution was mixedwith one microliter of the reservoir solution, which contains 15%polyethylenglycol 4000, 10% 2-Propanol, and 100 mM Citrate-Buffer pH5.6. Crystals were observed after one week.

The crystals obtained were washed with the reservoir solution. In thenext step, a single protein-ligand complex crystal was put into thereservoir solution, which contained in addition, a 10 mM TACE inhibitorto replace the co-crystallizedN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide. The crystal is usually kept for 72 hours in thesolution containing the inhibitor to allow the ligand replacement. Afterincubation, the crystal was frozen in liquid propane and used for X-raydiffraction.

Results

Two distinct structures of TACE:inhibitor complexes have been previouslydisclosed [Maskos et. al., Proc. Natl. Acad. Sci. USA 95:3408–3412(1998); Letavic et al. Biorgan. & Medic. Chem Lett. 12:1387–1390(2002)]. However, neither crystal appears to be amenable to crystalsoaking. As disclosed herein, it has been unexpectedly discovered thatthe uncomplexed TACE protein (apo-protein) is unstable at the highconcentrations required to grow and use crystals for X-raycrystallographic studies. Consistently, the crystal of Maskos et. al.has been found to be resistant to standard inhibitor soakingexperiments, severely limiting its value in structure based rationaldrug design.

Stability of TACE: The stability of TACE was examined under severalbuffer conditions at pH 7.5. Twenty microliter aliquots of TACE at 15mg/ml were desalted over P-6 spin columns (BioRAD, Hercules, Calif.)that had been equilibrated in:

(a) 25 mM Tris, 0.15M NaCl;

(b) 25 mM Tris; or

(c) 25 mM Tris plusN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide.

The stability of the TACE protein was evaluated after storage at 4degrees Celsius for seven days. After incubating the TACE polypeptideunder the three conditions listed above, Sodium Dodecyl SulfatePolyAcrylamide Gel Electrophoresis (SDS-PAGE) was performed. The resultsshow that only a single band having a molecular weight of 30 Kd wasobserved when either high salt (0.15 sodium chloride) or an inhibitor[N-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide, at a 1:1.5 molar ratio enzyme:inhibitor] wasincluded in the incubation. In direct contrast, two additional fainterbands that ran well ahead of the more significant TACE band wereobserved in the sample lacking either high salt or the inhibitor. Themolecular weights of these two additional fainter bands were 14 Kd and16 Kd, respectively, which add up to the 30 Kd molecular weight of theTACE polypeptide. N-terminal sequencing of the peptides corresponding tothe two additional bands indicated that they were indeed, proteolyticproducts of the TACE protein. Moreover, the sequencing data indicatedthe presence of a single cleavage site at 352Y–V353 of SEQ ID NO: 2.

Substrate Specificity of TACE: In an effort to understand the role ofthe different amino-acid residues of TACE regarding substratespecificity, a substitution study was performed at the P′1 position. Thecatalytic activity of TACE was determined in an assay using thesynthetic peptide Ac-SPLAQA-VRSSSR-NH2 (SEQ ID NO: 17) as the substrate.The sequence corresponds to the cleavage site of TACE on proTNF-alpha,with the sessile bond being between the alanine and the valine. Activitywas measured by incubating 100 nM TACE with 100 micromolar substrate in25 mM HEPES pH 7.3, 5 mM CaCl₂, for 1 hour at room temperature. Productformation was quantified at 214 mm after HPLC separation using aPOROS-R1 reverse phase column. Substitution of the P′1 valine (in boldabove) with either alanine, glycine or serine decreased activity of TACEto non-detectable levels. Based on this data it was decided tosubstitute the P′1 position of the internal cleave site [ . . .LGLAY-VGSPR . . . (SEQ ID NO: 16)] with one of these amino acids e.g.,either glycine or serine, in an attempt to eliminate theauto-proteolysis of TACE seen in the absence of NaCl.

Stability of TACE and TACE mutants: To test for stability underdifferent storage conditions, 20 ul of TACE protein, at 15 mg/ml, wasdesalted over BIO-RAD P-6 columns equilibrated at pH 7.5 in:

(a) 25 mM Tris, 0.15M NaCl;

(b) 25 mM Tris+1 mMN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide; or

(c) 25 mM Tris.

One microliter aliquots were analyzed by SDS-PAGE after storing them for3 hours, or 17 days at 4 degrees Celsius. In the absence of salt, thenative protein exhibits a pattern consistent with substantialproteolysis occurring after only 3 hours, with the protein beingcompletely proteolyzed after 17 days. In direct contrast, all constructswere stable in either 0.15M NaCl, or 1 mMN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide. The 2 loop mutants V353G and V353S showed improvedstability, with the V353G (vgTACE) mutant being highly resistant toauto-proteolysis, even after 17 days.

Crystallization. The TACE mutant V353G (vgTACE) could be crystallizedunder similar conditions as the native TACE [WO9940182, Published Aug.12, 1999, U.S. application Ser. No. 09/117,476, filed Jan. 27, 1999, thecontents of which are both hereby incorporated by reference in itsentirety]. vgTACE was concentrated to 15 mg/ml in 150 mM NaCl, 25 mMTris-HCl pH 8. After desalting the vgTACE with Bio-Rad P-6 columns,N-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide was added to a molecular ratio of 1:2(enzyme:inhibitor). The complex was crystallized using the hanging dropvapor diffusion technique. Equal amounts of vgTACE-inhibitor solutionwere mixed with the reservoir solution, containing 15% Polyethylenglycol4000, 10% 2-propanol, 100 mM sodium citrate pH 4.6, and equilibrated at295 degrees Kelvin. Crystals were observed after 7 days.

Crystallographic analysis: vgTACE crystals were washed using thereservoir solution. Glycerol was then added to the reservoir solution toa final concentration of 15%, and the crystals were flash frozen inliquid propane. X-ray diffraction data were collected at 100 degreesKelvin, using a rotating anode generator (Rigaku/MSC) or synchrotronsources. Diffraction was observed up to 1.7 Å. The vgTACE crystalsbelong to space group P2₁2₁2₁ (a=73, b=75, c=103 Å). There are twomolecules located within the asymmetric unit. Soaking with the compoundsof Table 2 was performed by incubation of the crystals in the reservoirsolution in the presence of up to 70 mM of the respective inhibitor. Inthe V353G mutant crystals, the co-crystallized inhibitorN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide could be replaced during soaking.

Model building and refinement: The crystal structure of V353G mutant wassolved by molecular replacement and refined using standardcrystallographic programs. The published TACE structure was used as thestarting model [PDB code:1BKC; Maskos et. al., Proc. Natl. Acad. Sci.USA 95:3408–3412 (1998)]. Replacement of the co-crystallized inhibitorwas verified by difference electron density maps. The vgTACE:inhibitorstructures were refined using X-PLOR(CNS). A list of vgTACE:inhibitorstructures that have been solved is shown in Table 2 below.

TABLE 2 RESOLUTION OF TACE-INHIBITOR COMPLEXES Resolution INHIBITOR(Angstroms) (4-biphenylsulfonyl)amino-propionic acid 1.73,4-dimethyl-2-(N-3'-pyridylmethyl-p- 1.9methoxysulfonamido)-benzenehydroxyamic acid alpha(R)-[[[4-2-butynyloxy)phenyl]sulfonyl]amino] 1.7 benzenepropanoic acidN-hydroxy-alpha(R),3(S)-dimethyl-3-[4-[(2-methyl-4- 2.1quinolinyl)methoxy]phenyl]-2-oxo-1-pyrrolidineacetamideN-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}- 2.0 Ile-Leu-OH

Table 3 below, comprises the coordinate set from the crystal structureof TACE complexed withN-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH. Toobtain these coordinates a single co-crystal of TACE in complex withN-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide was soaked in 10% polyethylenglycol 8000, 50 mMsodium citrate, 10 mM[N-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH] and10% dimethylsulphoxide (DMSO) for three days. The crystal wastransferred into a solution with additional 10% glycerol andflash-frozen in liquid nitrogen. X-ray diffraction data were collectedas described above. These data were processed and the structure wasrefined as described above.

The TACE monomer includes amino acid residues 6–259 of the catalyticdomain (see SEQ ID NO: 20) and is bound to the inhibitor,N-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH whichhas the number 260 in Table 3. The catalytic zinc ion has the number261. Amino acid residues Arg₂₈, Lys₇₂, Glu₈₁, Lys₈₈, Glu₉₃, Glu₉₄,Lys₉₅, and Arg₁₄₃ were modeled as Ala residues since their side chainswere disordered. The TACE protein used had the amino acid sequence ofSEQ ID NO: 20, i.e., Gly₁₃₉ is the single point mutation site replacingVal₁₃₉ of the TACE wild-type amino acid sequence.

In the data set below, one line contains information per one atom. Theseven columns of Table 3 represent respectively:

1) residue number,

2) one-letter amino acid code,

3) atom name,

4) x-coordinate,

5) y-coordinate,

6) z-coordinate, and

7) B-factor.

TABLE 3 COORDINATES for the COMPLEX of vgTACE withN-{3-(Hydroxyaminocarbonyl)-1-Oxo-(2R)-Benzylpropyl}-Ile-Leu-OH 6 PRO CB62.073 53.680 60.089 57.01 6 PRO CG 62.468 55.126 60.364 57.06 6 PRO C59.660 54.054 59.521 56.73 6 PRO O 59.094 53.151 60.132 56.72 6 PRO N61.472 55.081 58.226 56.90 6 PRO CD 62.545 55.773 58.963 57.18 6 PRO CA61.066 53.868 58.966 56.85 7 MET N 59.104 55.240 59.307 56.47 7 MET CA57.761 55.534 59.770 56.16 7 MET CB 57.653 57.002 60.180 57.02 7 MET CG58.514 57.370 61.375 58.06 7 MET SD 58.165 59.050 61.936 59.40 7 MET CE59.102 59.999 60.715 59.24 7 MET C 56.780 55.226 58.644 55.35 7 MET O55.612 54.925 58.885 55.64 8 LYS N 57.269 55.306 57.411 54.05 8 LYS CA56.458 55.020 56.236 52.67 8 LYS CB 56.923 55.885 55.062 53.02 8 LYS CG56.888 57.377 55.362 53.28 8 LYS CD 57.228 58.210 54.135 53.51 8 LYS CE57.057 59.698 54.425 53.52 8 LYS NZ 57.362 60.531 53.233 53.75 8 LYS C56.625 53.534 55.909 51.45 8 LYS O 57.436 53.153 55.066 51.21 9 ASN N55.843 52.701 56.585 49.93 9 ASN CA 55.927 51.263 56.398 48.31 9 ASN CB56.409 50.618 57.696 48.36 9 ASN CG 55.496 50.928 58.878 48.21 9 ASN OD155.835 50.643 60.029 48.47 9 ASN ND2 54.333 51.504 58.599 48.06 9 ASN C54.610 50.632 55.987 47.19 9 ASN O 54.456 49.415 56.074 47.11 10 THR N53.662 51.446 55.540 45.73 10 THR CA 52.367 50.913 55.151 44.26 10 THRCB 51.264 51.294 56.156 44.15 10 THR OG1 51.681 50.975 57.487 44.15 10THR CG2 49.992 50.519 55.834 43.84 10 THR C 51.883 51.361 53.787 43.3110 THR O 51.918 52.546 53.454 43.14 11 CYS N 51.420 50.393 53.007 42.0911 CYS CA 50.872 50.663 51.693 41.05 11 CYS C 49.373 50.626 51.927 40.7911 CYS O 48.813 49.571 52.249 40.43 11 CYS CB 51.310 49.574 50.705 40.4111 CYS SG 50.526 49.576 49.063 39.35 12 LYS N 48.737 51.792 51.812 40.4012 LYS CA 47.297 51.914 52.011 40.31 12 LYS CB 46.926 53.386 52.23640.50 12 LYS CG 47.582 54.002 53.467 40.84 12 LYS CD 47.193 53.24254.728 41.39 12 LYS CE 47.795 53.864 55.982 42.05 12 LYS NZ 47.30955.259 56.185 42.58 12 LYS C 46.567 51.353 50.793 40.16 12 LYS O 46.99651.573 49.660 39.93 13 LEU N 45.467 50.637 51.031 39.94 13 LEU CA 44.69850.015 49.954 39.87 13 LEU CB 44.540 48.506 50.189 39.57 13 LEU CG45.817 47.718 50.470 39.45 13 LEU CD1 45.455 46.287 50.855 39.64 13 LEUCD2 46.742 47.691 49.256 39.49 13 LEU C 43.283 50.536 49.735 40.21 13LEU O 42.590 50.941 50.674 40.15 14 LEU N 42.861 50.484 48.477 40.14 14LEU CA 41.506 50.825 48.098 40.45 14 LEU CB 41.451 51.674 46.823 40.2714 LEU CG 40.026 51.690 46.235 40.37 14 LEU CD1 39.053 52.388 47.19240.36 14 LEU CD2 39.992 52.378 44.869 40.24 14 LEU C 40.996 49.42847.775 40.61 14 LEU O 41.361 48.859 46.749 40.61 15 VAL N 40.186 48.85348.652 40.60 15 VAL CA 39.679 47.518 48.387 40.72 15 VAL CB 39.68546.670 49.668 40.68 15 VAL CG1 39.000 45.333 49.416 40.84 15 VAL CG241.133 46.454 50.110 40.43 15 VAL C 38.287 47.606 47.805 40.94 15 VAL O37.403 48.247 48.374 40.84 16 VAL N 38.104 46.979 46.648 41.32 16 VAL CA36.813 47.000 45.979 41.65 16 VAL CB 36.908 47.619 44.556 41.38 16 VALCG1 35.520 47.653 43.911 41.06 16 VAL CG2 37.502 49.030 44.633 41.07 16VAL C 36.218 45.610 45.844 42.07 16 VAL O 36.890 44.663 45.430 41.85 17ALA N 34.948 45.509 46.217 42.61 17 ALA CA 34.195 44.272 46.124 43.48 17ALA CB 33.525 43.956 47.466 43.31 17 ALA C 33.140 44.554 45.062 44.03 17ALA O 32.409 45.543 45.169 44.04 18 ASP N 33.073 43.714 44.032 44.77 18ASP CA 32.089 43.916 42.973 45.53 18 ASP CB 32.632 43.438 41.611 45.2018 ASP CG 32.781 41.928 41.520 45.15 18 ASP OD1 32.552 41.243 42.53445.18 18 ASP OD2 33.134 41.429 40.426 44.69 18 ASP C 30.798 43.19343.329 46.32 18 ASP O 30.702 42.584 44.398 46.49 19 HIS N 29.811 43.25542.440 47.07 19 HIS CA 28.519 42.626 42.699 47.79 19 HIS CB 27.50543.003 41.603 47.47 19 HIS CG 27.776 42.367 40.273 47.28 19 HIS CD227.273 41.247 39.704 47.04 19 HIS ND1 28.654 42.902 39.355 47.15 19 HISCE1 28.677 42.140 38.275 47.10 19 HIS NE2 27.848 41.129 38.462 47.20 19HIS C 28.585 41.109 42.835 48.24 19 HIS O 27.810 40.514 43.584 48.59 20ARG N 29.496 40.475 42.110 48.97 20 ARG CA 29.628 39.028 42.194 49.60 20ARG CB 30.652 38.517 41.180 50.00 20 ARG CG 30.268 38.661 39.709 50.3620 ARG CD 31.196 37.786 38.863 50.71 20 ARG NE 30.919 37.837 37.42850.98 20 ARG CZ 30.938 38.950 36.705 51.03 20 ARG NH1 31.217 40.11237.282 51.22 20 ARG NH2 30.693 38.900 35.404 51.10 20 ARG C 30.06438.606 43.601 49.92 20 ARG O 29.445 37.743 44.221 50.02 21 PHE N 31.13539.223 44.093 50.14 21 PHE CA 31.680 38.921 45.419 50.40 21 PHE CB32.929 39.773 45.671 49.84 21 PHE CG 33.724 39.353 46.875 49.29 21 PHECD1 34.687 38.353 46.773 49.02 21 PHE CD2 33.512 39.955 48.113 49.00 21PHE CE1 35.427 37.961 47.885 48.83 21 PHE CE2 34.247 39.567 49.227 48.8521 PHE CZ 35.206 38.568 49.112 48.76 21 PHE C 30.645 39.223 46.499 50.8021 PHE O 30.398 38.412 47.389 50.79 22 TYR N 30.057 40.410 46.406 51.4722 TYR CA 29.044 40.882 47.344 52.08 22 TYR CB 28.473 42.208 46.84752.14 22 TYR CG 27.346 42.764 47.688 52.33 22 TYR CD1 27.568 43.17649.002 52.41 22 TYR CE1 26.543 43.723 49.768 52.36 22 TYR CD2 26.06142.908 47.159 52.31 22 TYR CE2 25.027 43.451 47.915 52.30 22 TYR CZ25.274 43.857 49.219 52.34 22 TYR OH 24.260 44.396 49.980 52.15 22 TYR C27.902 39.890 47.527 52.49 22 TYR O 27.420 39.678 48.639 52.58 23 ARG N27.478 39.285 46.427 52.99 23 ARG CA 26.377 38.337 46.439 53.57 23 ARGCB 25.772 38.259 45.032 54.21 23 ARG CG 24.670 37.231 44.862 55.41 23ARG CD 24.194 37.165 43.414 56.15 23 ARG NE 23.138 36.171 43.243 57.1223 ARG CZ 22.521 35.920 42.092 57.61 23 ARG NH1 22.853 36.592 40.99357.83 23 ARG NH2 21.575 34.990 42.037 57.92 23 ARG C 26.757 36.93646.915 53.55 23 ARG O 26.147 36.400 47.841 53.56 24 TYR N 27.771 36.35246.288 53.52 24 TYR CA 28.201 34.997 46.617 53.49 24 TYR CB 28.80734.357 45.369 54.15 24 TYR CG 27.825 34.268 44.218 54.87 24 TYR CD126.763 33.362 44.252 55.28 24 TYR CE1 25.845 33.285 43.202 55.60 24 TYRCD2 27.945 35.100 43.103 55.19 24 TYR CE2 27.031 35.032 42.046 55.54 24TYR CZ 25.987 34.122 42.103 55.78 24 TYR OH 25.096 34.033 41.060 56.1624 TYR C 29.156 34.836 47.799 53.15 24 TYR O 29.227 33.763 48.393 53.1925 MET N 29.890 35.889 48.142 52.56 25 MET CA 30.820 35.816 49.263 51.9225 MET CB 32.197 36.350 48.850 51.12 25 MET CG 32.938 35.473 47.85650.04 25 MET SD 33.311 33.822 48.484 48.61 25 MET CE 34.533 34.18649.697 49.08 25 MET C 30.312 36.601 50.468 51.97 25 MET O 30.580 36.24451.610 51.92 26 GLY N 29.583 37.677 50.206 52.06 26 GLY CA 29.065 38.48951.289 52.32 26 GLY C 27.611 38.186 51.579 52.46 26 GLY O 26.975 38.88952.360 52.49 27 ARG N 27.089 37.137 50.949 52.57 27 ARG CA 25.698 36.74051.132 52.75 27 ARG CB 25.506 36.116 52.520 52.63 27 ARG C 24.799 37.96450.957 52.81 27 ARG O 23.786 38.121 51.642 52.84 28 GLY N 25.192 38.83150.028 52.75 28 GLY CA 24.429 40.032 49.748 52.70 28 GLY C 24.352 41.00250.906 52.59 28 GLY O 23.514 41.900 50.912 52.70 29 GLU N 25.225 40.83251.890 52.61 29 GLU CA 25.226 41.716 53.050 52.45 29 GLU CB 25.17840.900 54.340 52.72 29 GLU CG 24.292 39.673 54.284 53.26 29 GLU CD24.218 38.966 55.620 53.50 29 GLU OE1 23.661 39.561 56.564 53.95 29 GLUOE2 24.721 37.826 55.732 53.73 29 GLU C 26.462 42.614 53.079 52.17 29GLU O 27.596 42.152 52.911 52.18 30 GLU N 26.237 43.899 53.313 51.57 30GLU CA 27.325 44.854 53.365 51.00 30 GLU CB 26.760 46.264 53.525 51.3730 GLU CG 27.780 47.365 53.347 51.94 30 GLU CD 27.166 48.747 53.45152.20 30 GLU OE1 26.101 48.968 52.840 52.34 30 GLU OE2 27.751 49.61454.134 52.46 30 GLU C 28.278 44.539 54.516 50.33 30 GLU O 29.495 44.50554.338 50.25 31 SER N 27.717 44.300 55.697 49.48 31 SER CA 28.514 44.00556.880 48.61 31 SER CB 27.603 43.854 58.105 48.80 31 SER OG 26.74642.730 57.967 49.18 31 SER C 29.353 42.742 56.714 47.87 31 SER O 30.51642.710 57.105 47.78 32 THR N 28.755 41.707 56.134 46.96 32 THR CA 29.43640.435 55.924 46.18 32 THR CB 28.456 39.373 55.407 46.10 32 THR OG127.349 39.281 56.310 46.18 32 THR CG2 29.132 38.006 55.321 45.75 32 THRC 30.567 40.599 54.919 45.65 32 THR O 31.699 40.177 55.165 45.46 33 THRN 30.254 41.222 53.788 44.84 33 THR CA 31.248 41.451 52.752 44.08 33 THRCB 30.637 42.243 51.569 43.91 33 THR OG1 29.650 41.432 50.922 43.61 33THR CG2 31.710 42.623 50.550 43.80 33 THR C 32.436 42.211 53.333 43.6533 THR O 33.585 41.828 53.124 43.57 34 THR N 32.155 43.271 54.086 43.0934 THR CA 33.205 44.091 54.688 42.76 34 THR CB 32.614 45.317 55.41342.93 34 THR OG1 31.956 46.158 54.462 43.33 34 THR CG2 33.715 46.11856.100 43.26 34 THR C 34.082 43.328 55.676 42.38 34 THR O 35.314 43.43355.631 42.11 35 ASN N 33.449 42.566 56.564 41.68 35 ASN CA 34.183 41.80057.564 41.10 35 ASN CB 33.216 41.095 58.521 41.84 35 ASN CG 32.53442.065 59.473 42.72 35 ASN OD1 33.191 42.905 60.091 43.34 35 ASN ND231.212 41.952 59.600 43.12 35 ASN C 35.126 40.778 56.940 40.39 35 ASN O36.269 40.633 57.380 40.26 36 TYR N 34.647 40.075 55.920 39.29 36 TYR CA35.469 39.076 55.255 38.44 36 TYR CB 34.702 38.447 54.087 38.50 36 TYRCG 35.481 37.381 53.337 38.59 36 TYR CD1 36.460 37.729 52.406 38.87 36TYR CE1 37.201 36.751 51.737 38.98 36 TYR CD2 35.258 36.028 53.578 38.7636 TYR CE2 35.990 35.042 52.915 38.96 36 TYR CZ 36.960 35.412 51.99939.14 36 TYR OH 37.695 34.448 51.351 39.43 36 TYR C 36.755 39.728 54.75337.64 36 TYR O 37.852 39.220 54.987 37.54 37 LEU N 36.614 40.871 54.08936.62 37 LEU CA 37.764 41.582 53.544 35.66 37 LEU CB 37.293 42.67852.589 35.78 37 LEU CG 36.523 42.090 51.409 35.99 37 LEU CD1 35.93343.204 50.540 36.23 37 LEU CD2 37.423 41.175 50.572 35.84 37 LEU C38.692 42.170 54.592 34.90 37 LEU O 39.908 42.183 54.409 34.78 38 ILE N38.125 42.677 55.681 33.72 38 ILE CA 38.931 43.237 56.750 32.55 38 ILECB 38.045 43.881 57.838 32.75 38 ILE CG2 38.873 44.133 59.101 32.67 38ILE CG1 37.404 45.158 57.286 32.95 38 ILE CD1 36.421 45.792 58.267 33.0438 ILE C 39.764 42.117 57.380 32.11 38 ILE O 40.946 42.304 57.672 31.6339 GLU N 39.153 40.951 57.581 31.02 39 GLU CA 39.888 39.842 58.173 30.4839 GLU CB 38.920 38.752 58.652 30.75 39 GLU CG 38.105 39.226 59.85830.87 39 GLU CD 37.639 38.098 60.759 31.45 39 GLU OE1 38.342 37.06560.853 31.14 39 GLU OE2 36.576 38.263 61.402 31.89 39 GLU C 40.95739.272 57.236 29.76 39 GLU O 42.059 38.961 57.681 29.28 40 LEU N 40.63639.160 55.948 29.41 40 LEU CA 41.580 38.648 54.951 29.09 40 LEU CB40.910 38.551 53.568 28.82 40 LEU CG 41.832 38.033 52.437 28.74 40 LEUCD1 41.002 37.394 51.318 29.05 40 LEU CD2 42.690 39.160 51.834 28.74 40LEU C 42.793 39.581 54.869 29.05 40 LEU O 43.928 39.124 54.915 29.32 41ILE N 42.559 40.886 54.760 28.87 41 ILE CA 43.666 41.827 54.663 29.03 41ILE CG 43.176 43.251 54.326 29.55 41 ILE CG2 44.336 44.248 54.448 29.6141 ILE CG1 42.584 43.247 52.915 29.85 41 ILE CD1 43.620 42.893 51.87130.69 41 ILE C 44.505 41.843 55.920 28.81 41 ILE O 45.735 41.948 55.84328.95 42 ASP N 43.857 41.712 57.082 28.46 42 ASP CA 44.602 41.692 58.33428.44 42 ASP CB 43.642 41.680 59.532 28.61 42 ASP CG 44.364 41.53560.858 28.75 42 ASP OD1 45.085 42.463 61.281 29.10 42 ASP OD2 44.21140.477 61.477 29.27 42 ASP C 45.538 40.474 58.366 28.03 42 ASP O 46.68540.580 58.796 27.60 43 ARG N 45.063 39.324 57.898 28.15 43 ARG CA 45.90638.120 57.885 28.17 43 ARG CB 45.062 36.863 57.623 28.15 43 ARG CG44.122 36.454 58.770 27.76 43 ARG CD 43.472 35.089 58.475 27.71 43 ARGNE 42.820 35.046 57.161 26.80 43 ARG CZ 41.529 35.271 56.937 27.38 43ARG NH1 40.709 35.562 57.944 27.15 43 ARG NH2 41.048 35.194 55.696 27.0043 ARG C 47.046 38.202 56.857 27.98 43 ARG O 48.161 37.749 57.125 28.3644 VAL N 46.785 38.774 55.690 28.21 44 VAL CA 47.840 38.909 54.678 28.1844 VAL CB 47.269 39.445 53.341 28.21 44 VAL CG1 48.384 39.630 52.30827.55 44 VAL CG2 46.203 38.466 52.821 27.51 44 VAL C 48.878 39.87755.238 28.58 44 VAL O 50.096 39.657 55.139 28.66 45 ASP N 48.392 40.94055.865 28.68 45 ASP CA 49.279 41.922 56.450 28.96 45 ASP CB 48.46043.038 57.112 29.15 45 ASP CG 49.334 44.065 57.813 29.82 45 ASP OD150.246 44.634 57.167 29.51 45 ASP OD2 49.108 44.298 59.017 29.85 45 ASPC 50.207 41.239 57.462 29.03 45 ASP O 51.412 41.502 57.478 28.77 46 ASPN 49.660 40.348 58.292 29.37 46 ASP CA 50.483 39.637 59.281 29.71 46 ASPCB 49.672 38.556 60.019 29.76 46 ASP CG 48.718 39.133 61.080 30.25 46ASP OD1 48.876 40.305 61.490 29.71 46 ASP OD2 47.818 38.388 61.509 29.8746 ASP C 51.693 38.982 58.593 29.78 46 ASP O 52.816 39.029 59.108 29.6247 ILE N 51.460 38.382 57.427 30.23 47 ILE CA 52.538 37.735 56.679 30.6747 ILE CB 51.992 37.002 55.433 31.15 47 ILE CG2 53.125 36.727 54.44730.51 47 ILE CG1 51.279 35.721 55.872 31.48 47 ILE CD1 50.451 35.13254.766 32.09 47 ILE C 53.593 38.755 56.254 31.14 47 ILE O 54.792 38.51056.405 30.87 48 TYR N 53.148 39.896 55.732 31.49 48 TYR CA 54.068 40.94255.309 32.49 48 TYR CB 53.319 42.061 54.583 31.94 48 TYR CG 53.04041.766 53.135 31.45 48 TYR CD1 51.996 40.921 52.760 31.16 48 TYR CE151.788 40.592 51.431 31.44 48 TYR CD2 53.865 42.281 52.135 31.29 48 TYRCE2 53.663 41.959 50.801 31.23 48 TYR CZ 52.629 41.113 50.452 31.44 48TYR OH 52.432 40.774 49.131 31.26 48 TYR C 54.870 41.543 56.460 33.48 48TYR O 56.089 41.631 56.384 33.46 49 ARG N 54.185 41.952 57.526 34.66 49ARG CA 54.857 42.550 58.677 36.02 49 ARG CB 53.841 42.907 59.764 36.2649 ARG CG 52.763 43.847 59.289 36.68 49 ARG CD 53.292 45.229 58.93437.07 49 ARG NE 52.209 46.034 58.368 37.56 49 ARG CZ 52.193 47.36458.307 37.89 49 ARG NH1 53.213 48.077 58.781 37.88 49 ARG NH2 51.14047.983 57.785 37.74 49 ARG C 55.942 41.659 59.276 36.73 49 ARG O 57.01042.145 59.643 36.86 50 ASN N 55.674 40.359 59.373 37.87 50 ASN CA 56.64839.425 59.938 38.88 50 ASN CB 55.960 38.130 60.391 39.28 50 ASN CG55.127 38.313 61.653 40.06 50 ASN OD1 55.654 38.613 62.729 40.35 50 ASNND2 53.820 38.128 61.527 40.37 50 ASN C 57.785 39.076 58.981 39.41 50ASN O 58.719 38.361 59.355 39.55 51 THR N 57.716 39.570 57.748 39.83 51THR CA 58.759 39.273 56.766 40.21 51 THR CB 58.193 39.341 55.321 39.7151 THR OG1 57.247 38.284 55.130 39.05 51 THR CG2 59.311 39.214 54.28939.56 51 THR C 59.963 40.214 56.884 40.90 51 THR O 59.811 41.435 56.89840.72 52 ALA N 61.160 39.637 56.989 41.77 52 ALA CA 62.377 40.435 57.08142.50 52 ALA CB 63.324 39.846 58.129 42.80 52 ALA C 63.024 40.422 55.70243.07 52 ALA O 63.718 39.478 55.335 43.06 53 TRP N 62.779 41.481 54.94543.91 53 TRP CA 63.289 41.612 53.589 45.05 53 TRP CB 62.739 42.90052.974 44.35 53 TRP CG 61.263 43.028 53.191 44.14 53 TRP CD2 60.23742.245 52.570 43.92 53 TRP CE2 59.009 42.619 53.161 43.92 53 TRP CE360.236 41.260 51.574 43.79 53 TRP CD1 60.631 43.829 54.104 43.91 53 TRPNE1 59.280 43.587 54.094 43.79 53 TRP CZ2 57.790 42.039 52.790 43.71 53TRP CZ3 59.020 40.681 51.205 43.76 53 TRP CH2 57.817 41.076 51.813 43.6553 TRP C 64.812 41.568 53.469 46.12 53 TRP O 65.355 41.475 52.366 46.4054 ASP N 65.501 41.633 54.601 47.29 54 ASP CA 66.957 41.592 54.597 48.5954 ASP CB 67.516 42.909 55.136 48.87 54 ASP CG 67.313 43.057 56.62449.36 54 ASP OD1 66.354 42.460 57.157 49.66 54 ASP OD2 68.106 43.77757.264 49.88 54 ASP C 67.405 40.438 55.481 49.24 54 ASP O 68.549 40.38755.914 49.44 55 ASN N 66.486 39.514 55.748 50.07 55 ASN CA 66.766 38.35556.590 50.93 55 ASN CB 67.749 37.415 55.888 51.38 55 ASN CG 67.17636.824 54.618 51.78 55 ASN OD1 66.118 36.188 54.642 52.49 55 ASN ND267.866 37.028 53.500 51.87 55 ASN C 67.325 38.768 57.948 51.31 55 ASN O67.796 37.932 58.719 51.50 56 ALA N 67.259 40.062 58.238 51.62 56 ALA CA67.760 40.590 59.497 51.85 56 ALA CB 68.875 41.606 59.230 51.88 56 ALA C66.641 41.237 60.306 51.99 56 ALA O 65.756 40.550 60.827 52.12 57 GLY N66.683 42.562 60.407 52.02 57 GLY CA 65.673 43.273 61.172 51.90 57 GLY C64.834 44.247 60.371 51.78 57 GLY O 64.105 45.066 60.941 51.80 58 PHE N64.934 44.170 59.047 51.52 58 PHE CA 64.161 45.056 58.192 51.17 58 PHECB 64.863 45.257 56.850 51.44 58 PHE CG 64.329 46.414 56.053 51.66 58PHE CD1 64.497 47.719 56.504 51.71 58 PHE CD2 63.679 46.201 54.838 51.8258 PHE CE1 64.030 48.797 55.754 51.85 58 PHE CE2 63.206 47.273 54.07851.76 58 PHE CZ 63.383 48.573 54.536 51.92 58 PHE C 62.791 44.427 57.97850.89 58 PHE O 62.525 43.813 56.945 50.82 59 LYS N 61.933 44.565 58.98150.35 59 LYS CA 60.584 44.027 58.924 49.76 59 LYS CB 60.455 42.83559.875 50.20 59 LYS CG 60.515 43.213 61.340 50.70 59 LYS CD 61.26342.182 62.176 51.68 59 LYS CE 60.698 40.771 62.023 52.16 59 LYS NZ61.171 40.095 60.774 52.55 59 LYS C 59.623 45.130 59.342 49.05 59 LYS O60.017 46.292 59.468 49.13 60 GLY N 58.366 44.768 59.563 48.23 60 GLY CA57.391 45.757 59.973 47.17 60 GLY C 56.658 46.411 58.821 46.43 60 GLY O55.847 47.304 59.041 46.28 61 TYR N 56.945 45.979 57.594 45.82 61 TYR CA56.274 46.529 56.421 45.19 61 TYR CB 57.231 46.588 55.220 45.23 61 TYRCG 58.318 47.628 55.362 45.61 61 TYR CD1 59.495 47.344 56.054 45.69 61TYR CE1 60.476 48.310 56.228 46.00 61 TYR CD2 58.150 48.913 54.844 45.7261 TYR CE2 59.128 49.891 55.017 46.07 61 TYR CZ 60.287 49.579 55.71146.07 61 TYR OH 61.254 50.534 55.898 46.61 61 TYR C 55.030 45.713 56.05044.60 61 TYR O 55.018 44.486 56.151 44.29 62 GLY N 53.982 46.404 55.61744.24 62 GLY CA 52.764 45.710 55.245 43.54 62 GLY C 51.743 46.578 54.54743.26 62 GLY O 52.071 47.584 53.916 43.26 63 ILE N 50.486 46.178 54.67442.96 63 ILE CA 49.382 46.884 54.055 42.58 63 ILE CB 48.708 45.99952.994 42.40 63 ILE CG2 49.671 45.795 51.825 42.19 63 ILE CG1 48.28344.674 53.628 42.25 63 ILE CD1 47.610 43.744 52.647 42.44 63 ILE C48.350 47.298 55.096 42.70 63 ILE O 48.451 46.940 56.265 42.36 64 GLN N47.353 48.053 54.655 42.99 64 GLN CA 46.312 48.535 55.549 43.58 64 GLNCB 46.872 49.646 56.435 43.72 64 GLN CG 46.027 49.964 57.643 44.56 64GLN CD 46.544 51.166 58.404 44.90 64 GLN OE1 46.220 52.311 58.076 45.0664 GLN NE2 47.369 50.915 59.417 45.02 64 GLN C 45.185 49.080 54.69243.80 64 GLN O 45.424 49.864 53.774 43.54 65 ILE N 43.960 48.654 54.97344.15 65 ILE CA 42.821 49.133 54.208 44.77 65 ILE CB 41.545 48.35854.549 44.68 65 ILE CG2 40.369 48.967 53.790 44.65 65 ILE CG1 41.74246.876 54.225 44.77 65 ILE CD1 40.501 46.043 54.484 44.90 65 ILE C42.612 50.611 54.517 45.39 65 ILE O 42.644 51.032 55.673 45.02 66 GLU N42.424 51.395 53.466 46.36 66 GLU CA 42.217 52.825 53.603 47.29 66 GLUCB 43.081 53.570 52.595 47.91 66 GLU CG 42.907 55.071 52.625 48.97 66GLU CD 44.110 55.764 53.218 49.62 66 GLU OE1 44.403 55.552 54.422 50.0766 GLU OE2 44.774 56.516 52.469 49.95 66 GLU C 40.757 53.128 53.33647.78 66 GLU O 40.147 53.964 54.001 47.77 67 GLN N 40.211 52.445 52.34248.29 67 GLN CA 38.820 52.613 51.971 49.15 67 GLN CB 38.660 53.77350.981 49.40 67 GLN CG 37.210 54.029 50.600 50.14 67 GLN CD 37.02555.165 49.609 50.48 67 GLN OE1 35.895 55.527 49.284 50.88 67 GLN NE238.127 55.729 49.120 50.48 67 GLN C 38.304 51.330 51.334 49.40 67 GLN O39.037 50.640 50.621 49.36 68 ILE N 37.044 51.010 51.604 49.87 68 ILE CA36.419 49.822 51.042 50.50 68 ILE CB 35.922 48.853 52.135 50.51 68 ILECG2 35.176 47.695 51.472 50.47 68 ILE CG1 37.096 48.365 52.984 50.62 68ILE CD1 36.657 47.442 54.122 50.63 68 ILE C 35.222 50.236 50.203 51.1168 ILE O 34.411 51.054 50.625 51.20 69 ARG N 35.115 49.672 49.010 51.8169 ARG CA 34.001 49.973 48.132 52.54 69 ARG CB 34.488 50.628 46.84053.05 69 ARG CG 35.030 52.025 47.035 53.82 69 ARG CD 34.029 52.90147.769 54.59 69 ARG NE 34.453 54.296 47.791 55.44 69 ARG CZ 34.49755.078 46.715 55.75 69 ARG NH1 34.140 54.601 45.528 55.91 69 ARG NH234.897 56.340 46.823 55.87 69 ARG C 33.268 48.694 47.807 52.75 69 ARG O33.845 47.762 47.256 52.71 70 ILE N 31.992 48.651 48.162 53.11 70 ILE CA31.177 47.479 47.898 53.49 70 ILE CB 30.459 47.005 49.183 53.40 70 ILECG2 29.657 45.738 48.885 53.34 70 ILE CG1 31.484 46.782 50.296 53.46 70ILE CD1 30.841 46.428 51.613 53.40 70 ILE C 30.138 47.815 46.839 53.8570 ILE O 29.202 48.566 47.099 53.84 71 LEU N 30.317 47.271 45.639 54.3771 LEU CA 29.379 47.506 44.549 54.89 71 LEU CB 30.055 47.275 43.19854.74 71 LEU CG 31.388 48.015 43.074 54.71 71 LEU CD1 32.007 47.76841.699 54.69 71 LEU CD2 31.210 49.515 43.310 54.57 71 LEU C 28.22946.526 44.719 55.34 71 LEU O 28.326 45.368 44.307 55.27 72 LYS N 27.14447.002 45.324 55.90 72 LYS CA 25.969 46.177 45.587 56.55 72 LYS CB24.938 46.986 46.376 56.47 72 LYS C 25.323 45.575 44.343 56.94 72 LYS O24.900 44.421 44.357 56.97 73 SER N 25.245 46.346 43.265 57.51 73 SER CA24.637 45.839 42.038 58.13 73 SER CB 23.242 46.451 41.846 58.24 73 SEROG 23.283 47.867 41.883 58.49 73 SER C 25.497 46.099 40.805 58.43 73 SERO 26.317 47.018 40.790 58.35 74 PRO N 25.313 45.287 39.750 58.80 74 PROCD 24.286 44.237 39.626 58.89 74 PRO CA 26.069 45.414 38.501 59.17 74PRO CB 25.339 44.466 37.553 59.11 74 PRO CG 24.798 43.424 38.464 58.9574 PRO C 26.069 46.838 37.976 59.58 74 PRO O 25.188 47.630 38.310 59.5475 GLN N 27.067 47.157 37.159 60.04 75 GLN CA 27.183 48.482 36.565 60.4375 GLN CB 28.652 48.859 36.377 60.42 75 GLN CG 28.870 50.163 35.61960.40 75 GLN CD 28.245 51.355 36.315 60.49 75 GLN OE1 28.527 51.62437.480 60.43 75 GLN NE2 27.389 52.080 35.599 60.54 75 GLN C 26.49548.464 35.211 60.80 75 GLN O 26.787 47.614 34.374 60.79 76 GLU N 25.57449.397 35.001 61.39 76 GLU CA 24.859 49.474 33.732 61.84 76 GLU CB23.699 50.466 33.841 62.11 76 GLU CG 22.626 50.078 34.855 62.52 76 GLUCD 21.468 51.068 34.878 62.88 76 GLU OE1 20.799 51.224 33.830 63.21 76GLU OE2 21.227 51.690 35.937 62.90 76 GLU C 25.830 49.924 32.643 62.0076 GLU O 26.533 50.918 32.811 62.07 77 VAL N 25.871 49.192 31.534 62.2077 VAL CA 26.770 49.531 30.435 62.49 77 VAL CB 27.862 48.459 30.25762.52 77 VAL CG1 28.693 48.369 31.537 62.62 77 VAL CG2 27.224 47.10829.924 62.51 77 VAL C 26.085 49.736 29.083 62.64 77 VAL O 25.424 48.83928.544 62.75 78 LYS N 26.270 50.930 28.535 62.74 78 LYS CA 25.698 51.29127.248 62.72 78 LYS CB 26.135 52.715 26.895 62.87 78 LYS CG 25.96653.683 28.068 63.12 78 LYS CD 26.509 55.069 27.760 63.35 78 LYS CE25.682 55.757 26.695 63.52 78 LYS NZ 26.220 57.097 26.322 63.67 78 LYS C26.164 50.296 26.184 62.54 78 LYS O 27.154 49.584 26.382 62.53 79 PRO N25.440 50.216 25.051 62.36 79 PRO CD 24.173 50.912 24.749 62.31 79 PROCA 25.796 49.298 23.964 62.20 79 PRO CB 24.878 49.745 22.832 62.24 79PRO CG 23.626 50.111 23.578 62.28 79 PRO C 27.273 49.375 23.580 61.91 79PRO O 27.788 50.452 23.270 62.02 80 GLY N 27.947 48.228 23.605 61.55 80GLY CA 29.356 48.175 23.258 60.89 80 GLY C 30.309 48.534 24.385 60.40 80GLY O 31.483 48.166 24.331 60.45 81 GLU N 29.819 49.255 25.391 59.82 81GLU CA 30.642 49.675 26.526 59.12 81 GLU CB 29.969 50.851 27.250 59.1681 GLU C 30.926 48.540 27.518 58.67 81 GLU O 30.174 47.562 27.603 58.6982 LYS N 32.021 48.683 28.262 57.81 82 LYS CA 32.429 47.696 29.263 57.0182 LYS CB 33.671 46.933 28.809 57.13 82 LYS CG 33.473 46.000 27.63757.26 82 LYS CD 34.827 45.557 27.082 57.37 82 LYS CE 35.623 46.74326.528 57.56 82 LYS NZ 36.890 46.328 25.854 57.55 82 LYS C 32.756 48.34430.597 56.39 82 LYS O 33.085 49.530 30.678 56.15 83 HIS N 32.668 47.53531.643 55.70 83 HIS CA 32.969 47.970 32.999 55.05 83 HIS CB 31.76748.705 33.606 54.78 83 HIS CG 32.061 49.374 34.916 54.53 83 HIS CD232.328 50.664 35.218 54.59 83 HIS ND1 32.126 48.682 36.107 54.54 83 HISCE1 32.419 49.520 37.085 54.44 83 HIS NE2 32.548 50.730 36.574 54.55 83HIS C 33.291 46.701 33.787 54.71 83 HIS O 32.661 45.662 33.575 54.68 84TYR N 34.276 46.768 34.679 54.17 84 TYR CA 34.645 45.587 35.455 53.76 84TYR CB 35.851 45.880 36.350 52.91 84 TYR CG 35.579 46.812 37.510 52.2584 TYR CD1 35.627 48.199 37.351 51.75 84 TYR CE1 35.431 49.059 38.43851.53 84 TYR CD2 35.316 46.303 38.785 51.78 84 TYR CE2 35.113 47.15039.876 51.52 84 TYR CZ 35.175 48.522 39.701 51.42 84 TYR OH 35.00749.343 40.789 50.97 84 TYR C 33.493 45.059 36.310 53.81 84 TYR O 33.43743.867 36.617 53.72 85 ASN N 32.571 45.941 36.680 54.04 85 ASN CA 31.43745.550 37.511 54.28 85 ASN CB 31.208 46.599 38.604 54.07 85 ASN CG30.175 46.160 39.630 53.85 85 ASN OD1 30.173 45.012 40.072 53.62 85 ASNND2 29.302 47.081 40.026 53.78 85 ASN C 30.133 45.308 36.746 54.72 85ASN O 29.061 45.282 37.347 54.66 86 MET N 30.222 45.121 35.430 55.28 86MET CA 29.032 44.865 34.622 55.89 86 MET CB 29.305 45.136 33.133 56.0286 MET CG 30.326 44.225 32.476 56.14 86 MET SD 30.704 44.761 30.77556.77 86 MET CE 30.752 43.196 29.895 56.07 86 MET C 28.568 43.425 34.81356.27 86 MET O 29.360 42.545 35.169 56.28 87 ALA N 27.283 43.191 34.56456.60 87 ALA CA 26.688 41.871 34.735 56.88 87 ALA CB 25.177 41.95334.505 56.95 87 ALA C 27.285 40.784 33.855 57.08 87 ALA O 27.508 39.66234.310 57.04 88 LYS N 27.542 41.107 32.595 57.40 88 LYS CA 28.097 40.12331.676 57.94 88 LYS CB 27.616 40.413 30.251 57.93 88 LYS C 29.621 40.08931.715 58.25 88 LYS O 30.272 41.107 31.956 58.25 89 SER N 30.187 38.91231.489 58.63 89 SER CA 31.633 38.785 31.476 59.25 89 SER CB 32.05937.355 31.823 59.17 89 SER OG 31.725 36.450 30.787 59.39 89 SER C 32.07039.146 30.062 59.60 89 SER O 31.258 39.140 29.136 59.66 90 TYR N 33.34439.475 29.900 59.97 90 TYR CA 33.884 39.847 28.597 60.31 90 TYR CB34.038 41.371 28.537 60.49 90 TYR CG 34.554 41.917 27.225 60.75 90 TYRCD1 35.901 41.812 26.881 60.73 90 TYR CE1 36.377 42.321 25.672 60.86 90TYR CD2 33.691 42.545 26.326 60.76 90 TYR CE2 34.156 43.056 25.117 60.8190 TYR CZ 35.498 42.942 24.796 60.86 90 TYR OH 35.957 43.457 23.60460.88 90 TYR C 35.238 39.163 28.438 60.43 90 TYR O 35.975 39.017 29.41160.46 91 PRO N 35.597 38.746 27.209 60.56 91 PRO CD 36.958 38.218 27.00060.61 91 PRO CA 34.888 38.845 25.925 60.69 91 PRO CB 36.017 38.71924.915 60.67 91 PRO CG 36.899 37.713 25.577 60.65 91 PRO C 33.772 37.83025.645 60.74 91 PRO O 32.830 38.138 24.908 60.86 92 ASN N 33.877 36.62726.208 60.72 92 ASN CA 32.862 35.594 25.989 60.66 92 ASN CB 33.48234.202 26.115 60.91 92 ASN CG 32.607 33.117 25.509 61.25 92 ASN OD131.443 32.951 25.889 61.45 92 ASN ND2 33.164 32.372 24.557 61.35 92 ASNC 31.701 35.713 26.970 60.49 92 ASN O 31.734 35.126 28.051 60.44 93 GLUN 30.674 36.463 26.581 60.23 93 GLU CA 29.502 36.672 27.427 59.90 93 GLUCB 28.433 37.469 26.667 60.10 93 GLU C 28.911 35.362 27.919 59.61 93 GLUO 28.273 35.322 28.974 59.58 94 GLU N 29.140 34.289 27.166 59.18 94 GLUCA 28.603 32.976 27.518 58.67 94 GLU CB 28.631 32.061 26.289 58.81 94GLU C 29.302 32.291 28.696 58.28 94 GLU O 28.686 31.486 29.405 58.33 95LYS N 30.579 32.604 28.900 57.63 95 LYS CA 31.352 32.013 29.986 56.88 95LYS CB 32.843 32.047 29.640 56.89 95 LYS C 31.102 32.719 31.323 56.39 95LYS O 30.639 33.863 31.368 56.43 96 ASP N 31.415 32.026 32.414 55.64 96ASP CA 31.230 32.568 33.758 54.84 96 ASP CB 31.501 31.479 34.805 55.1996 ASP CG 31.613 32.036 36.220 55.58 96 ASP OD1 30.665 32.704 36.68555.84 96 ASP OD2 32.650 31.798 36.868 55.61 96 ASP C 32.100 33.78234.073 53.98 96 ASP O 31.639 34.734 34.707 53.84 97 ALA N 33.348 33.75833.616 52.96 97 ALA CA 34.270 34.845 33.916 51.86 97 ALA CB 35.35334.325 34.866 52.03 97 ALA C 34.934 35.558 32.742 51.02 97 ALA O 35.05635.020 31.638 50.90 98 TRP N 35.373 36.784 33.014 49.95 98 TRP CA 36.08037.600 32.036 48.91 98 TRP CB 36.458 38.962 32.608 48.72 98 TRP CG35.441 40.023 32.645 48.66 98 TRP CD2 35.581 41.334 32.094 48.56 98 TRPCE2 34.458 42.081 32.503 48.57 98 TRP CE3 36.556 41.953 31.296 48.57 98TRP CD1 34.264 40.019 33.335 48.50 98 TRP NE1 33.671 41.253 33.260 48.3998 TRP CZ2 34.279 43.421 32.145 48.50 98 TRP CZ3 36.379 43.284 30.94148.55 98 TRP CH2 35.248 44.004 31.367 48.57 98 TRP C 37.413 36.93931.772 48.06 98 TRP O 37.813 36.005 32.470 47.91 99 ASP N 38.104 37.45330.761 47.24 99 ASP CA 39.457 37.013 30.468 46.10 99 ASP CB 39.92937.579 29.137 46.43 99 ASP CG 41.427 37.508 28.986 46.46 99 ASP OD141.941 36.400 28.727 47.06 99 ASP OD2 42.092 38.550 29.144 46.60 99 ASPC 40.144 37.776 31.600 45.23 99 ASP O 40.003 39.002 31.691 44.88 100 VALN 40.867 37.073 32.466 44.29 100 VAL CA 41.502 37.725 33.605 43.47 100VAL CB 42.310 36.706 34.465 43.18 100 VAL CG1 43.509 36.157 33.689 42.91100 VAL CG2 42.758 37.388 35.760 42.85 100 VAL C 42.387 38.923 33.25643.23 100 VAL O 42.304 39.971 33.909 42.97 101 LYS N 43.225 38.77732.233 42.98 101 LYS CA 44.107 39.864 31.822 43.00 101 LYS CB 45.03339.393 30.690 43.37 101 LYS CG 46.058 40.425 30.248 43.86 101 LYS CD46.966 39.889 29.141 44.34 101 LYS CE 47.942 40.968 28.673 44.98 101 LYSNZ 49.056 40.417 27.830 44.94 101 LYS C 43.281 41.079 31.377 42.76 101LYS O 43.573 42.216 31.754 42.74 102 MET N 42.245 40.838 30.579 42.66102 MET CA 41.385 41.924 30.121 42.38 102 MET CB 40.387 41.424 29.07742.94 102 MET CG 41.000 41.193 27.696 43.54 102 MET SD 39.812 40.66326.435 44.26 102 MET CE 40.520 39.136 25.937 44.13 102 MET C 40.63242.531 31.296 41.87 102 MET O 40.461 43.744 31.373 42.04 103 LEU N40.186 41.687 32.220 41.39 103 LEU CA 39.457 42.177 33.376 40.80 103 LEUCB 38.980 41.007 34.245 40.97 103 LEU CG 38.421 41.464 35.605 41.14 103LEU CD1 37.354 42.554 35.429 41.16 103 LEU CD2 37.840 40.278 36.37640.92 103 LEU C 40.315 43.124 34.207 40.26 103 LEU O 39.854 44.18134.630 39.94 104 LEU N 41.570 42.751 34.432 39.97 104 LEU CA 42.45743.586 35.234 39.64 104 LEU CG 43.802 42.873 35.453 39.38 104 LEU CG44.769 43.686 36.322 39.11 104 LEU CD1 44.122 44.066 37.656 38.99 104LEU CD2 46.064 42.905 36.567 39.19 104 LEU C 42.673 44.958 34.591 39.70104 LEU O 42.686 45.977 35.282 39.74 105 GLU N 42.829 44.991 33.27039.72 105 GLU CA 43.038 46.262 32.590 39.81 105 GLU CB 43.403 46.04831.113 39.50 105 GLU CG 44.044 47.290 30.505 39.45 105 GLU CD 44.53347.106 29.080 39.30 105 GLU OE1 45.020 46.004 28.735 39.29 105 GLU OE244.446 48.082 28.308 39.37 105 GLU C 41.783 47.130 32.701 40.04 105 GLUO 41.875 48.339 32.914 39.95 106 GLN N 40.611 46.510 32.565 40.23 106GLN CA 39.351 47.246 32.676 40.32 106 GLN CB 38.159 46.328 32.436 40.79106 GLN CG 36.847 47.083 32.344 41.31 106 GLN CD 36.810 47.977 31.12241.76 106 GLN OE1 36.925 47.497 30.003 42.24 106 GLN NE2 36.663 49.28231.331 41.92 106 GLN C 39.214 47.853 34.066 40.23 106 GLN O 38.88949.037 34.225 39.87 107 PHE N 39.451 47.030 35.080 39.95 107 PHE CA39.350 47.502 36.448 39.81 107 PHE CB 39.753 46.396 37.425 39.46 107 PHECG 39.855 46.859 38.843 39.13 107 PHE CD1 38.759 47.434 39.479 38.92 107PHE CD2 41.057 46.745 39.541 39.16 107 PHE CE1 38.853 47.892 40.78338.80 107 PHE CE2 41.166 47.200 40.852 38.87 107 PHE CZ 40.062 47.77741.475 39.11 107 PHE C 40.249 48.714 36.635 39.97 107 PHE O 39.85249.705 37.249 39.64 108 SER N 41.467 48.624 36.100 40.20 108 SER CA42.435 49.708 36.198 40.65 108 SER CB 43.764 49.281 35.555 40.75 108 SEROG 44.397 48.270 36.326 40.70 108 SER C 41.901 50.976 35.525 41.00 108SER O 42.091 52.085 36.023 40.78 109 PHE N 41.232 50.796 34.392 41.58109 PHE CA 40.664 51.914 33.647 42.31 109 PHE CB 40.138 51.422 32.29442.55 109 PHE CG 39.381 52.468 31.512 43.19 109 PHE CD1 40.058 53.48930.840 43.31 109 PHE CD2 37.986 52.444 31.466 43.13 109 PHE CE1 39.35854.472 30.134 43.43 109 PHE CE2 37.277 53.421 30.766 43.47 109 PHE CZ37.965 54.440 30.097 43.28 109 PHE C 39.530 52.604 34.409 42.61 109 PHEO 39.489 53.828 34.504 42.49 110 ASP N 38.616 51.810 34.955 43.11 110ASP CA 37.468 52.356 35.672 43.65 110 ASP CB 36.394 51.278 35.834 43.73110 ASP CG 35.826 50.827 34.511 43.66 110 ASP OD1 35.467 51.703 33.69844.35 110 ASP OD2 35.725 49.605 34.280 43.87 110 ASP C 37.756 52.98537.026 44.03 110 ASP O 37.168 54.011 37.367 44.05 111 ILE N 38.65852.384 37.796 44.24 111 ILE CA 38.982 52.903 39.118 44.73 111 ILE CB39.388 51.739 40.074 44.96 111 ILE CG2 40.899 51.509 40.001 45.08 111ILE CG1 38.920 52.040 41.499 45.06 111 ILE CD1 37.407 52.123 41.60545.37 111 ILE C 40.097 53.952 39.091 44.84 111 ILE O 40.495 54.47240.134 44.79 112 ALA N 40.581 54.269 37.894 45.28 112 ALA CA 41.66855.234 37.703 45.60 112 ALA CB 41.664 55.721 36.251 45.60 112 ALA C41.703 56.437 38.656 46.11 112 ALA O 42.695 56.654 39.359 45.85 113 GLUN 40.633 57.230 38.672 46.58 113 GLU CA 40.579 58.404 39.542 47.25 113GLU CB 39.206 59.003 39.453 48.10 113 GLU CG 38.831 59.503 38.044 49.39113 GLU CD 38.447 58.322 37.168 50.14 113 GLU OE1 38.460 58.476 35.92750.83 113 GLU OE2 38.124 57.243 37.721 50.63 113 GLU C 40.882 58.05840.996 47.10 113 GLU O 41.702 58.714 41.640 46.96 114 GLU N 40.22357.028 41.515 47.26 114 GLU CA 40.459 56.624 42.895 47.33 114 GLU CB39.307 55.624 43.339 48.04 114 GLU CG 37.978 56.204 43.279 49.07 114 GLUCD 36.938 55.295 43.903 49.76 114 GLU OE1 37.024 55.045 45.129 50.01 114GLU OE2 36.034 54.831 43.169 50.18 114 GLU C 41.859 56.027 43.058 46.96114 GLU O 42.580 56.374 43.990 46.83 115 ALA N 42.250 55.155 42.13146.60 115 ALA CA 43.560 54.518 42.188 46.18 115 ALA CB 43.763 53.63640.954 46.08 115 ALA C 44.692 55.540 42.296 45.96 115 ALA O 45.73255.261 42.888 45.88 116 SER N 44.479 56.726 41.734 45.83 116 SER CA45.491 57.781 41.752 45.45 116 SER CB 45.051 58.941 40.857 45.79 116 SEROG 43.950 59.637 41.431 46.56 116 SER C 45.783 58.321 43.145 45.01 116SER O 46.820 58.942 43.370 44.95 117 LYS N 44.877 58.078 44.084 44.49117 LYS CA 45.034 58.592 45.438 43.96 117 LYS CB 43.699 59.167 45.91644.62 117 LYS CG 43.180 60.294 45.040 45.26 117 LYS CD 41.749 60.68245.391 45.71 117 LYS CE 41.290 61.840 44.512 46.27 117 LYS NZ 39.90562.270 44.841 46.64 117 LYS C 45.562 57.617 46.481 43.42 117 LYS O45.813 58.014 47.617 43.44 118 VAL N 45.735 56.351 46.111 42.52 118 VALCA 46.239 55.359 47.062 41.47 118 VAL CB 45.155 54.330 47.435 41.77 118VAL CG1 44.181 54.939 48.444 41.78 118 VAL CG2 44.417 53.882 46.16841.66 118 VAL C 47.457 54.579 46.589 40.74 118 VAL O 47.823 54.61145.416 40.46 119 CYS N 48.076 53.867 47.526 39.88 119 CYS CA 49.24253.057 47.226 38.75 119 CYS C 48.871 51.928 46.269 38.23 119 CYS O49.550 51.705 45.262 37.93 119 CYS CB 49.828 52.489 48.516 38.99 119 CYSSG 51.156 51.304 48.189 39.08 120 LEU N 47.782 51.232 46.582 37.29 120LEU CA 47.294 50.130 45.758 36.83 120 LEU CB 47.825 48.787 46.268 36.78120 LEU CG 49.289 48.477 45.935 36.84 120 LEU CD1 49.699 47.143 46.58436.69 120 LEU CD2 49.505 48.411 44.416 36.93 120 LEU C 45.776 50.04445.758 36.55 120 LEU O 45.117 50.448 46.712 36.38 121 ALA N 45.23749.498 44.676 36.37 121 ALA CA 43.802 49.283 44.539 36.19 121 ALA CB43.233 50.126 43.398 36.11 121 ALA C 43.670 47.790 44.225 35.94 121 ALAO 44.370 47.278 43.354 35.76 122 HIS N 42.789 47.092 44.937 35.85 122HIS CA 42.614 45.661 44.717 35.65 122 HIS CB 43.191 44.869 45.903 35.42122 HIS CG 43.523 43.447 45.575 35.09 122 HIS CD2 42.833 42.302 45.77834.99 122 HIS ND1 44.674 43.086 44.906 35.06 122 HIS CE1 44.675 41.78044.710 34.72 122 HIS NE2 43.571 41.280 45.227 34.70 122 HIS C 41.14745.298 44.532 35.95 122 HIS O 40.281 45.718 45.306 35.96 123 LEU N40.882 44.503 43.503 36.17 123 LEU CA 39.536 44.053 43.186 36.35 123 LEUCB 39.325 44.126 41.676 36.14 123 LEU CG 38.054 43.424 41.186 36.51 123LEU CD1 36.797 44.062 41.781 35.94 123 LEU CD2 37.995 43.461 39.65235.81 123 LEU C 39.250 42.627 43.668 36.77 123 LEU O 39.970 41.68543.334 36.61 124 PHE N 38.204 42.482 44.480 36.97 124 PHE CA 37.79841.166 44.955 37.31 124 PHE CB 37.563 41.158 46.470 36.56 124 PHE CG38.827 41.275 47.275 35.87 124 PHE CD1 39.350 42.520 47.601 35.46 124PHE CD2 39.510 40.130 47.683 35.68 124 PHE CE1 40.536 42.630 48.32735.35 124 PHE CE2 40.701 40.225 48.411 35.04 124 PHE CZ 41.211 41.47648.732 35.19 124 PHE C 36.515 40.847 44.222 37.97 124 PHE O 35.51041.533 44.395 37.89 125 THR N 36.576 39.819 43.387 38.53 125 THR CA35.442 39.382 42.589 39.62 125 THR CB 35.743 39.519 41.082 39.80 125 THROG1 34.580 09.180 40.313 40.27 125 THR CG2 36.882 38.572 40.698 40.00125 THR C 35.143 37.914 42.872 40.18 125 THR O 35.818 37.267 43.67339.87 126 TYR N 34.129 37.392 42.197 40.95 126 TYR CA 33.747 35.99842.363 41.69 126 TYR CB 32.552 35.879 43.318 41.98 126 TYR CG 32.25934.454 43.736 42.49 126 TYR CD1 33.053 33.808 44.685 42.57 126 TYR CE132.818 32.482 45.039 42.99 126 TYR CD2 31.218 33.735 43.151 42.74 126TYR CE2 30.973 32.403 43.495 42.81 126 TYR CZ 31.777 31.784 44.437 42.91126 TYR OH 31.566 30.465 44.755 43.11 126 TYR C 33.394 35.384 41.00842.12 126 TYR O 32.224 35.230 40.670 42.34 127 GLN N 34.415 35.06540.219 42.76 127 GLN CA 34.208 34.442 38.915 43.26 127 GLN CB 34.21735.498 37.799 43.14 127 GLN CG 35.246 36.612 37.946 42.91 127 GLN CD34.958 37.792 37.024 42.59 127 GLN OE1 34.938 37.654 35.803 42.66 127GLN NE2 34.735 38.958 37.610 42.61 127 GLN C 35.260 33.360 38.672 43.75127 GLN O 36.400 33.474 39.120 43.51 128 ASP N 34.861 32.303 37.97344.39 128 ASP CA 35.745 31.179 37.705 45.26 128 ASP CB 34.920 29.89337.597 46.00 128 ASP CG 35.770 28.639 37.682 46.60 128 ASP OD1 37.01128.752 37.811 47.14 128 ASP OD2 35.186 27.533 37.626 47.15 128 ASP C36.548 31.386 36.434 45.56 128 ASP O 36.023 31.228 35.331 45.53 129 PHEN 37.822 31.740 36.602 45.62 129 PHE CA 38.719 31.982 35.477 45.80 129PHE CB 39.944 32.797 35.916 45.38 129 PHE CG 39.615 34.179 36.412 44.95129 PHE CD1 38.936 35.081 35.599 44.72 129 PHE CD2 39.986 34.578 37.69044.59 129 PHE CE1 38.630 36.362 36.057 44.77 129 PHE CE2 39.687 35.85438.158 44.62 129 PHE CZ 39.006 36.750 37.342 44.61 129 PHE C 39.19330.673 34.875 46.19 129 PHE O 39.149 29.622 35.521 46.15 130 ASP N39.651 30.755 33.630 46.61 130 ASP CA 40.154 29.597 32.896 47.04 130 ASPCB 40.314 29.949 31.417 47.59 130 ASP CG 39.089 29.600 30.604 48.18 130ASP OD1 38.725 28.403 30.574 48.64 130 ASP OD2 38.490 30.514 29.99448.61 130 ASP C 41.479 29.056 33.412 46.87 130 ASP O 42.295 29.79233.967 46.99 131 MET N 41.682 27.759 33.211 46.72 131 MET CA 42.90727.088 33.618 46.61 131 MET CB 44.112 27.686 32.873 47.71 131 MET CG43.962 27.784 31.348 49.21 131 MET SD 45.510 28.252 30.493 50.81 131 METCE 45.787 29.965 31.114 50.14 131 MET C 43.155 27.148 35.129 45.94 131MET O 44.265 26.870 35.582 45.93 132 GLY N 42.132 27.517 35.899 44.94132 GLY CA 42.277 27.580 37.346 44.07 132 GLY C 43.000 28.794 37.91443.19 132 GLY O 43.528 28.745 39.033 43.33 133 THR N 43.023 29.88237.150 42.18 133 THR CA 43.666 31.121 37.578 40.98 133 THR CB 43.80832.099 36.383 41.19 133 THR OG1 44.649 71.515 35.377 41.40 133 THR CG244.414 33.427 36.842 41.23 133 THR C 42.809 31.771 38.674 39.94 133 THRO 41.586 31.842 38.546 39.84 134 LEU N 43.444 32.239 39.747 38.76 134LEU CA 42.711 32.869 40.845 37.81 134 LEU CB 43.042 32.173 42.175 37.94134 LEU CG 42.389 30.789 42.318 37.96 134 LEU CD1 42.685 30.206 43.69837.70 134 LEU CD2 40.870 30.873 42.122 37.86 134 LEU C 42.927 34.37741.003 37.01 134 LEU O 42.134 35.065 41.650 36.89 135 GLY N 43.99834.902 40.430 36.22 135 GLY CA 44.235 36.328 40.559 35.22 135 GLY C45.271 36.830 39.581 34.68 135 GLY O 45.902 36.035 38.886 34.19 136 LEUN 45.449 38.146 39.530 34.01 136 LEU CA 46.423 38.747 38.625 33.85 136LEU CB 45.798 38.886 37.229 33.98 136 LEU CG 46.809 39.145 36.108 34.21136 LEU CD1 47.681 37.906 35.869 34.29 136 LEU CD2 46.072 39.523 34.81433.73 136 LEU C 46.806 40.120 39.182 33.49 136 LEU O 45.999 40.76639.852 33.44 137 ALA N 48.032 40.563 38.912 33.26 137 ALA CA 48.50141.858 39.402 33.14 137 ALA CB 48.891 41.743 40.876 32.82 137 ALA C49.691 42.373 38.603 33.33 137 ALA O 50.378 41.597 37.942 32.89 138 TYRN 49.932 43.679 38.679 33.35 138 TYR CA 51.051 44.300 37.967 34.13 138TYR CB 50.633 45.642 37.343 33.72 138 TYR CG 49.420 45.578 36.435 34.20138 TYR CD1 49.409 44.750 35.311 34.16 138 TYR CE1 48.314 44.718 34.44434.48 138 TYR CD2 48.295 46.380 36.676 34.16 138 TYR CE2 47.192 46.35935.812 34.13 138 TYR CZ 47.209 45.526 34.700 34.39 138 TYR OH 46.12945.474 33.843 34.66 138 TYR C 52.214 44.554 38.924 34.55 138 TYR O52.031 44.643 40.139 34.22 139 GLY N 53.411 44.688 38.360 35.47 139 GLYCA 54.582 44.949 39.173 36.62 139 GLY C 55.701 43.955 38.966 37.31 139GLY O 56.866 44.278 39.201 37.52 140 GLY N 55.364 42.753 38.509 38.05140 GLY CA 56.390 41.745 38.301 39.17 140 GLY C 56.480 41.123 36.91539.93 140 GLY O 56.986 40.010 36.772 39.75 141 SER N 56.005 41.83035.892 40.71 141 SER CA 56.047 41.310 34.521 41.34 141 SER CB 54.72640.612 34.171 41.61 141 SER OG 53.644 41.531 34.107 41.68 141 SER C56.315 42.424 33.510 41.80 141 SER O 55.864 43.556 33.690 41.68 142 PRON 57.042 42.112 32.418 42.10 142 PRO CD 57.531 43.131 31.468 42.24 142PRO CA 57.606 40.797 32.089 42.58 142 PRO CB 58.156 41.011 30.681 42.47142 PRO CG 58.675 42.413 30.770 42.16 142 PRO C 58.695 40.313 33.05342.97 142 PRO O 59.102 39.148 33.000 43.23 143 ARG N 59.167 41.20433.922 43.15 143 ARG CA 60.210 40.849 34.876 43.34 143 ARG CB 61.58641.240 34.315 43.34 143 ARG C 59.975 41.523 36.230 43.27 143 ARG O59.069 42.353 36.380 43.08 144 ALA N 60.792 41.161 37.217 43.16 144 ALACA 60.658 41.746 38.540 43.21 144 ALA CB 61.685 41.136 39.500 43.24 144ALA C 60.846 43.258 38.457 43.31 144 ALA O 61.550 43.759 37.578 43.14145 ASN N 60.197 43.968 39.377 43.45 145 ASN CA 60.261 45.422 39.47243.71 145 ASN CB 61.657 45.857 39.936 43.64 145 ASN CG 62.076 45.18341.237 43.64 145 ASN OD1 62.790 44.185 41.227 43.65 145 ASN ND2 61.61645.720 42.359 43.62 145 ASN C 59.892 46.151 38.183 43.96 145 ASN O60.412 47.228 37.904 44.11 146 SER N 58.986 45.568 37.406 44.30 146 SERCA 58.561 46.178 36.149 44.53 146 SER CB 58.053 45.095 35.194 44.90 146SER OG 57.994 45.580 33.861 45.57 146 SER C 57.468 47.231 36.390 44.52146 SER O 57.213 47.616 37.536 44.02 147 HIS N 56.838 47.693 35.30644.59 147 HIS CA 55.772 48.698 35.377 44.76 147 HIS CB 55.110 48.91334.014 45.09 147 HIS CG 56.026 49.448 32.966 45.61 147 HIS CD2 56.44150.712 32.701 45.76 147 HIS ND1 56.620 48.642 32.022 45.97 147 HIS CE157.360 49.383 31.216 46.05 147 HIS NE2 57.266 50.642 31.607 45.97 147HIS C 54.667 48.317 36.349 44.59 147 HIS O 54.346 47.137 36.509 44.78148 GLY N 54.072 49.323 36.978 44.20 148 GLY CA 52.992 49.056 37.90543.92 148 GLY C 53.439 48.668 39.297 43.71 148 GLY O 54.623 48.74639.643 43.63 149 GLY N 52.481 48.226 40.101 43.39 149 GLY CA 52.79847.862 41.461 43.23 149 GLY C 52.524 49.052 42.354 43.19 149 GLY O51.878 50.010 41.938 42.72 150 VAL N 53.028 48.994 43.576 43.52 150 VALCA 52.813 50.047 44.557 44.13 150 VAL CB 53.640 49.776 45.836 43.94 150VAL CG1 53.225 48.426 46.429 43.80 150 VAL CG2 55.142 49.794 45.51243.74 150 VAL C 53.109 51.464 44.072 44.80 150 VAL O 53.981 51.68543.233 44.70 151 CYS N 52.360 52.416 44.621 45.70 151 CYS CA 52.49053.833 44.301 46.52 151 CYS C 51.926 54.161 42.928 46.62 151 CYS O52.421 53.688 41.910 46.64 151 CYS CB 53.952 54.272 44.383 47.29 151 CYSSG 54.701 54.094 46.035 48.62 152 PRO N 50.881 54.996 42.885 46.88 152PRO CD 50.302 55.789 43.988 46.91 152 PRO CA 50.275 55.360 41.608 47.02152 PRO CB 49.057 56.178 42.030 47.05 152 PRO CG 49.553 56.888 43.24846.98 152 PRO C 51.227 56.152 40.715 47.22 152 PRO O 51.923 57.05841.170 47.34 153 LYS N 51.271 55.768 39.449 47.40 153 LYS CA 52.08356.441 38.445 47.63 153 LYS CB 53.417 55.730 38.211 48.04 153 LYS CG54.161 56.286 36.997 48.80 153 LYS CD 55.514 55.622 36.766 49.22 153 LYSCE 56.049 55.963 35.379 49.72 153 LYS NZ 56.112 57.435 35.118 49.77 153LYS C 51.225 56.360 37.199 47.58 153 LYS O 50.798 55.275 36.804 47.34154 ALA N 50.955 57.506 36.588 47.43 154 ALA CA 50.107 57.533 35.41147.58 154 ALA CB 49.631 58.970 35.148 47.52 154 ALA C 50.751 56.95934.156 47.66 154 ALA O 51.945 57.135 33.912 47.36 155 TYR N 49.93556.245 33.385 47.93 155 TYR CA 50.332 55.663 32.108 48.40 155 TYR CB50.496 54.145 32.178 48.59 155 TYR CG 51.659 53.666 33.007 48.89 155 TYRCD1 51.577 53.627 34.396 49.04 155 TYR CE1 52.648 53.180 35.172 49.22155 TYR CD2 52.846 53.246 32.400 49.00 155 TYR CE2 53.923 52.797 33.16649.11 155 TYR CZ 53.815 52.765 34.553 49.11 155 TYR OH 54.867 52.31335.317 48.98 155 TYR C 49.156 55.981 31.201 48.65 155 TYR O 48.02055.591 31.489 48.52 156 TYR N 49.422 56.695 30.115 48.94 156 TYR CA48.362 57.068 29.196 49.32 156 TYR CB 48.830 58.192 28.261 49.84 156 TYRCG 47.712 58.733 27.392 50.40 156 TYR CD1 46.759 59.608 27.915 50.55 156TYR CE1 45.697 60.059 27.141 50.83 156 TYR CD2 47.573 58.322 26.06450.63 156 TYR CE2 46.511 58.763 25.283 50.88 156 TYR CZ 45.579 59.62925.827 50.96 156 TYR OH 44.519 60.052 25.059 51.42 156 TYR C 47.88655.884 28.368 49.30 156 TYR O 48.671 55.239 27.673 49.46 157 SER N46.595 55.595 28.453 49.12 157 SER CA 46.010 54.509 27.688 49.10 157 SERCB 44.894 53.839 28.489 48.89 157 SER OG 44.305 52.779 27.759 48.59 157SER C 45.448 55.103 26.395 49.27 157 SER O 44.431 55.794 26.412 49.40158 PRO N 46.126 54.865 25.259 49.31 158 PRO CD 47.451 54.230 25.13849.24 158 PRO CA 45.680 55.382 23.959 49.35 158 PRO CB 46.646 54.72122.986 49.33 158 PRO CG 47.923 54.721 23.781 49.38 158 PRO C 44.22555.043 23.668 49.29 158 PRO O 43.462 55.901 23.228 49.38 159 VAL N43.841 53.796 23.918 49.12 159 VAL CA 42.464 53.380 23.693 49.15 159 VALCB 42.311 51.848 23.780 49.15 159 VAL CG1 40.836 51.468 23.636 49.23 159VAL CG2 43.119 51.183 22.673 49.17 159 VAL C 41.543 54.018 24.736 49.15159 VAL O 40.398 54.357 24.445 49.18 160 GLY N 42.049 54.178 25.95349.05 160 GLY CA 41.242 54.771 27.005 48.98 160 GLY C 41.118 56.27926.901 48.70 160 GLY O 40.251 56.880 27.537 48.86 161 LYS N 41.97956.887 26.094 48.39 161 LYS CA 41.984 58.331 25.914 48.06 161 LYS CB40.707 58.782 25.197 48.57 161 LYS CG 40.574 58.210 23.792 49.22 161 LYSCD 39.228 58.548 23.165 49.84 161 LYS CE 39.116 57.933 21.776 50.37 161LYS NZ 37.765 58.122 21.153 50.81 161 LYS C 42.113 59.041 27.252 47.59161 LYS O 41.632 60.163 27.417 47.67 162 LYS N 42.763 58.384 28.21046.75 162 LYS CA 42.962 58.973 29.531 46.11 162 LYS CB 41.665 58.93730.349 46.11 162 LYS CG 41.400 57.609 31.058 46.38 162 LYS CD 40.27757.753 32.073 46.41 162 LYS CE 40.057 56.480 32.873 46.71 162 LYS NZ38.867 56.602 33.778 46.17 162 LYS C 44.042 58.230 30.300 45.48 162 LYSO 44.426 57.128 29.929 45.34 163 ASN N 44.533 58.839 31.371 44.81 163ASN CA 45.544 58.185 32.184 44.41 163 ASN CB 46.232 59.171 33.120 44.52163 ASN CG 47.001 60.220 32.380 44.76 163 ASN OD1 47.576 59.951 31.32845.24 163 ASN ND2 47.032 61.425 32.926 44.78 163 ASN C 44.912 57.09033.022 43.96 163 ASN O 43.740 57.160 33.399 43.97 164 ILE N 45.70256.066 33.299 43.24 164 ILE CA 45.249 54.961 34.114 42.52 164 ILE CB44.841 53.761 33.239 42.41 164 ILE CG2 43.707 54.206 32.318 42.46 164ILE CG1 46.040 53.223 32.447 42.23 164 ILE CD1 46.908 52.260 33.24641.87 164 ILE C 46.433 54.627 34.999 42.09 164 ILE O 47.530 55.15734.794 41.98 165 TYR N 46.216 53.777 35.993 41.37 165 TYR CA 47.30053.410 36.881 40.63 165 TYR CB 47.073 54.025 38.268 41.16 165 TYR CG46.943 55.538 38.225 41.76 165 TYR CD1 45.740 56.145 37.852 42.03 165TYR CE1 45.632 57.531 37.738 42.27 165 TYR CD2 48.040 56.361 38.48841.97 165 TYR CE2 47.946 57.754 38.373 42.23 165 TYR CZ 46.735 58.32837.995 42.58 165 TYR OH 46.628 59.693 37.847 42.79 165 TYR C 47.43951.896 36.943 39.77 165 TYR O 46.458 51.157 36.790 39.50 166 LEU N48.671 51.442 37.143 38.73 166 LEU CA 48.959 50.015 37.202 37.71 166 LEUCB 50.128 49.689 36.266 37.45 166 LEU CG 49.830 50.047 34.805 37.43 166LEU CD1 51.057 49.796 33.921 37.38 166 LEU CD2 48.640 49.243 34.28337.14 166 LEU C 49.286 49.560 38.621 37.06 166 LEU O 49.983 48.56638.819 36.54 167 ASN N 48.799 50.312 39.604 36.57 167 ASN CA 49.01349.979 41.005 36.02 167 ASN CB 49.129 51.264 41.835 35.90 167 ASN CG47.866 52.122 41.782 35.90 167 ASN OD1 47.078 52.047 40.835 35.38 167ASN ND2 47.682 52.952 42.800 35.49 167 ASN C 47.783 49.171 41.411 35.89167 ASN O 47.042 49.547 42.314 35.95 168 SER N 47.582 48.042 40.74235.61 168 SER CA 46.409 47.231 41.009 35.49 168 SER CB 45.273 47.71540.124 35.83 168 SER OG 45.729 47.807 38.786 36.17 168 SER C 46.56345.736 40.813 35.19 168 SER O 47.544 45.248 40.247 34.75 169 GLY N45.548 45.023 41.285 34.71 169 GLY CA 45.515 43.584 41.179 34.72 169 GLYC 44.141 43.073 41.582 34.64 169 GLY O 43.335 43.816 42.145 34.48 170LEU N 43.855 41.814 41.281 34.68 170 LEU CA 42.561 41.251 41.645 34.73170 LEU CB 41.607 41.284 40.439 34.66 170 LEU CG 42.086 40.441 39.24934.68 170 LEU CD1 41.551 39.014 39.336 34.63 170 LEU CD2 41.609 41.06337.932 35.09 170 LEU C 42.673 39.833 42.189 34.65 170 LEU O 43.63539.103 41.916 34.21 171 THR N 41.668 39.460 42.969 34.78 171 THR CA41.590 38.147 43.579 35.11 171 THR CB 41.803 38.211 45.115 34.88 171 THROG1 43.161 38.556 45.414 34.61 171 THR CG2 41.444 36.868 45.755 34.57171 THR C 40.192 37.606 43.344 35.57 171 THR O 39.211 38.342 43.44435.84 172 SER N 40.101 36.323 43.023 36.15 172 SER CA 38.806 35.68442.821 36.77 172 SER CB 38.679 35.115 41.405 36.76 172 SER OG 37.49034.342 41.298 36.64 172 SER C 38.729 34.548 43.827 37.20 172 SER O39.711 33.830 44.031 37.07 173 THR N 37.578 34.390 44.471 37.77 173 THRCA 37.430 33.325 45.448 38.35 173 THR CB 36.824 33.867 46.756 38.23 173THR OG1 35.577 34.511 46.480 37.93 173 THR CG2 37.787 34.877 47.39637.78 173 THR C 36.581 32.165 44.915 39.17 173 THR O 36.047 31.36345.689 38.97 174 LYS N 36.463 32.082 43.590 39.81 174 LYS CA 35.71731.006 42.948 40.76 174 LYS CB 34.533 31.558 42.149 40.83 174 LYS CG33.657 30.477 41.497 41.18 174 LYS CD 32.508 31.079 40.709 41.38 174 LYSCE 31.724 30.009 39.943 41.88 174 LYS NZ 30.543 30.600 39.218 42.08 174LYS C 36.664 30.275 42.006 41.39 174 LYS O 37.503 30.896 41.359 41.31175 ASN N 36.530 28.957 41.932 42.11 175 ASN CA 37.382 28.158 41.05942.96 175 ASN CB 38.770 27.983 41.691 42.77 175 ASN CG 39.808 27.47940.698 42.84 175 ASN OD1 39.721 27.752 39.501 42.69 175 ASN ND2 40.80826.759 41.198 42.67 175 ASN C 36.711 26.807 40.848 43.46 175 ASN O36.223 26.195 41.801 43.65 176 TYR N 36.685 26.353 39.595 44.06 176 TYRCA 36.055 25.083 39.245 44.41 176 TYR CB 36.945 23.909 39.676 45.08 176TYR CG 38.335 23.920 39.054 45.89 176 TYR CD1 38.525 23.666 37.688 46.26176 TYR CE1 39.810 23.693 37.120 46.51 176 TYR CD2 39.459 24.199 39.83046.18 176 TYR CE2 40.736 24.229 39.270 46.46 176 TYR CZ 40.903 23.97837.923 46.52 176 TYR OH 42.169 24.035 37.392 47.33 176 TYR C 34.67825.000 39.914 44.40 176 TYR O 34.361 24.045 40.626 44.47 177 GLY N33.884 26.045 39.704 44.31 177 GLY CA 32.536 26.102 40.244 43.91 177 GLYC 32.340 26.213 41.750 43.76 177 GLY O 31.208 26.389 42.206 43.81 178LYS N 33.415 26.122 42.526 43.32 178 LYS CA 33.296 26.194 43.987 42.94178 LYS CB 33.913 24.934 44.617 43.40 178 LYS CG 33.138 23.658 44.31744.05 178 LYS CD 33.875 22.393 44.750 44.34 178 LYS CE 33.049 21.15744.389 44.84 178 LYS NZ 33.794 19.866 44.501 45.03 178 LYS C 33.94027.427 44.614 42.19 178 LYS O 34.745 28.114 43.985 42.28 179 THR N33.548 27.719 45.850 41.39 179 THR CA 34.142 28.824 46.584 40.46 179 THRCB 33.265 29.251 47.765 40.32 179 THR OG1 32.033 29.780 47.269 40.47 179THR CG2 33.972 30.317 48.605 40.08 179 THR C 35.431 28.203 47.113 39.85179 THR O 35.396 27.107 47.666 39.95 180 ILE N 36.563 28.877 46.92938.94 180 ILE CA 37.839 28.327 47.385 38.10 180 ILE CB 39.037 29.04646.706 37.98 180 ILE CG2 38.852 28.980 45.187 37.87 180 ILE CG1 39.15230.489 47.215 37.90 180 ILE CD1 40.253 31.299 46.509 37.67 180 ILE C37.952 28.479 48.893 37.44 180 ILE O 37.200 29.230 49.495 37.34 181 LEUN 38.889 27.763 49.496 36.91 181 LEU CA 39.090 27.836 50.940 36.32 181LEU CB 40.127 26.792 51.373 36.29 181 LEU CG 39.647 25.347 51.160 36.15181 LEU CD1 40.729 24.351 51.572 36.24 181 LEU CD2 38.370 25.075 51.97036.93 181 LEU C 39.559 29.236 51.333 35.93 181 LEU O 40.141 29.95150.519 35.92 182 THR N 39.287 29.636 52.572 35.30 182 THR CA 39.71930.939 53.054 34.71 182 THR CB 39.209 31.195 54.488 35.13 182 THR OG137.778 31.307 54.471 35.39 182 THR CG2 39.823 32.483 55.047 35.65 182THR C 41.255 90.998 53.045 34.02 182 THR O 41.846 32.004 52.650 33.73183 LYS N 41.899 29.917 53.472 33.23 183 LYS CA 43.353 29.876 53.49732.54 183 LYS CB 43.843 28.583 54.154 32.61 183 LYS CG 43.545 27.31553.371 32.68 183 LYS CD 44.198 26.114 54.023 32.79 183 LYS CE 44.14724.900 53.111 33.20 183 LYS NZ 44.906 23.737 53.680 33.32 183 LYS C43.948 30.004 52.089 32.34 183 LYS O 45.078 30.476 51.936 31.99 184 GLUN 43.198 29.574 51.071 31.88 184 GLU CA 43.667 29.676 49.682 31.79 184GLU CB 42.859 28.751 48.753 32.16 184 GLU CG 42.872 27.283 49.166 32.95184 GLU CD 41.977 26.407 48.287 33.41 184 GLU OE1 40.936 26.893 47.82533.91 184 GLU OE2 42.315 25.233 48.072 34.31 184 GLU C 43.503 31.12649.239 31.34 184 GLU O 44.401 31.701 48.610 31.32 185 ALA N 42.36831.723 49.596 30.75 185 ALA CA 42.088 33.121 49.249 30.21 185 ALA CB40.708 33.523 49.772 30.06 185 ALA C 43.172 34.045 49.830 29.95 185 ALAO 43.603 34.997 49.183 29.57 186 ASP N 43.598 33.769 51.062 29.71 186ASP CA 44.654 34.553 51.695 29.29 186 ASP CB 44.970 34.007 53.092 29.21186 ASP CG 43.869 34.291 54.106 29.44 186 ASP OD1 42.860 34.927 53.75128.72 186 ASP OD2 44.029 33.870 55.268 29.64 186 ASP C 45.927 34.47750.856 29.17 186 ASP O 46.666 35.455 50.721 28.88 187 LEU N 46.19233.295 50.316 29.08 187 LEU CA 47.386 33.079 49.511 29.71 187 LEU CB47.648 31.564 49.358 30.11 187 LEU CG 47.946 30.921 50.728 31.10 187 LEUCD1 48.287 29.441 50.587 31.30 187 LEU CD2 49.106 31.640 51.419 31.18187 LEU C 47.315 33.783 48.150 29.27 187 LEU O 48.301 34.350 47.70029.26 188 VAL N 46.151 33.768 47.512 29.33 188 VAL CA 45.991 34.43846.226 28.96 188 VAL CB 44.530 34.290 45.694 29.41 188 VAL CG1 44.37935.028 44.356 29.10 188 VAL CG2 44.186 32.805 45.503 29.21 188 VAL C46.321 35.929 46.410 28.70 188 VAL O 47.158 36.507 45.697 28.11 189 TSRN 45.691 36.537 47.404 27.95 189 THR CA 45.910 37.949 47.670 27.87 189THR CB 44.899 38.444 48.725 28.07 189 THR OG1 43.580 38.244 48.213 27.14189 THR CG2 45.083 39.940 48.998 27.79 189 TSR C 47.349 38.302 48.08627.47 189 THR O 47.881 39.326 47.650 27.36 190 THR N 47.971 37.47348.920 27.06 190 THR CA 49.349 37.721 49.356 26.95 190 THR CB 49.85536.607 50.310 26.97 190 THR OG1 49.024 36.535 51.472 26.25 190 THR CG251.306 36.879 50.734 26.12 190 THR C 50.251 37.709 48.114 27.31 190 THRO 51.143 38.541 47.962 27.08 191 HIS N 49.988 36.747 47.234 27.47 191HIS CA 50.740 36.558 45.991 27.77 191 HIS CB 50.303 35.248 45.326 27.41191 HIS CG 50.934 34.995 43.990 27.50 191 HIS CD2 50.646 35.485 42.75527.59 191 HIS ND1 51.950 34.083 43.810 27.34 191 HIS CE1 52.259 74.01342.525 27.71 191 HIS NE2 51.479 34.853 41.865 27.92 191 HIS C 50.55937.717 45.002 27.83 191 HIS O 51.530 38.199 44.423 28.20 192 GLU N49.322 38.147 44.790 27.77 192 GLU CA 49.086 39.251 43.876 27.96 192 GLUCB 47.588 39.432 43.627 28.01 192 GLU CG 46.898 38.207 43.019 28.59 192GLU CD 47.664 37.557 41.865 29.60 192 GLU OE1 48.456 38.259 41.177 29.97192 GLU OE2 47.452 36.337 41.634 29.61 192 GLU C 49.723 40.529 44.43927.98 192 GLU O 50.335 41.307 43.687 27.71 193 LEU N 49.594 40.74245.751 27.68 193 LEU CA 50.207 41.908 46.376 27.69 193 LEU CB 49.81342.035 47.854 27.93 193 LEU CG 48.343 42.445 48.037 28.23 193 LEU CD147.999 42.597 49.525 28.74 193 LEU CD2 48.052 43.752 47.305 28.07 193LEU C 51.721 41.788 46.261 27.81 193 LEU O 52.419 42.799 46.182 27.59194 GLY N 52.214 40.548 46.261 27.45 194 GLY CA 53.640 40.309 46.12727.67 194 GLY C 54.145 40.860 44.805 27.89 194 GLY O 55.214 41.48444.758 27.74 195 HIS N 53.387 40.618 43.730 28.34 195 HIS CA 53.72841.138 42.409 28.91 195 HIS CB 52.722 40.684 41.344 28.90 195 HIS CG52.885 39.265 40.904 29.19 195 HIS CD2 51.972 38.284 40.707 29.13 195HIS ND1 54.107 38.728 40.555 29.27 195 HIS CE1 53.940 37.475 40.16729.41 195 HIS NE2 52.653 37.181 40.252 29.67 195 HIS C 53.686 42.66842.479 29.47 195 HIS O 54.517 43.357 41.876 29.42 196 ASN N 52.70243.197 43.201 29.77 196 ASN CA 52.554 44.642 43.350 30.47 196 ASN CB51.306 44.963 44.180 30.42 196 ASN CG 50.025 44.913 43.364 30.22 196 ASNOD1 48.930 44.822 43.919 30.73 196 ASN ND2 50.154 44.984 42.047 30.10196 ASN C 53.784 45.215 44.033 30.97 196 ASN O 54.210 46.326 43.72631.07 197 PHE N 54.332 44.460 44.983 31.16 197 PHE CA 55.527 44.89345.683 32.11 197 PHE CB 55.654 44.211 47.056 32.06 197 PHE CG 54.89744.909 48.160 32.49 197 PHE CD1 53.505 44.971 48.142 32.55 197 PHE CD255.575 45.481 49.233 32.76 197 PHE CE1 52.797 45.586 49.173 32.63 197PHE CE2 54.876 46.102 50.275 32.94 197 PHE CZ 53.479 46.154 50.243 32.84197 PHE C 56.770 44.604 44.839 32.36 197 PHE O 57.880 44.755 45.31732.90 198 GLY N 56.566 44.169 43.597 32.88 198 GLY CA 57.672 43.91342.683 33.15 198 GLY C 58.218 42.507 42.461 33.54 198 GLY O 59.08442.313 41.595 33.69 199 ALA N 57.752 41.522 43.219 33.46 199 ALA CA58.275 40.172 43.038 33.97 199 ALA CB 57.988 39.314 44.278 33.62 199 ALAC 57.723 39.486 41.802 34.11 199 ALA O 56.575 39.721 41.399 33.95 200GLU N 58.567 38.668 41.173 34.47 200 GLU CA 58.142 37.884 40.021 34.84200 GLU CB 59.177 37.931 38.876 36.00 200 GLU CG 60.553 37.335 39.15637.49 200 GLU CD 61.565 37.624 38.028 38.44 200 GLU OE1 61.245 37.38136.848 38.87 200 GLU OE2 62.680 38.093 38.330 39.55 200 GLU C 58.02436.483 40.626 34.62 200 GLU O 58.230 36.312 41.827 33.71 201 HIS N57.687 35.489 39.819 34.34 201 HIS CA 57.537 34.142 40.346 34.53 201 HISCB 56.856 33.266 39.299 33.54 201 HIS CG 55.433 33.644 39.047 32.67 201HIS CD2 54.541 34.303 39.824 32.28 201 HIS ND1 54.770 33.331 37.88132.61 201 HIS CE1 53.530 33.783 37.946 32.40 201 HIS NE2 53.366 34.37739.116 32.32 201 HIS C 58.842 33.498 40.796 35.26 201 HIS O 59.93333.867 40.341 35.22 202 ASP N 58.727 32.559 41.728 36.11 202 ASP CA59.894 31.828 42.187 36.98 202 ASP CB 59.589 31.079 43.488 36.36 202 ASPCG 59.438 32.010 44.670 35.88 202 ASP OD1 60.198 32.988 44.737 35.54 202ASP OD2 58.575 31.767 45.534 35.71 202 ASP C 60.210 30.824 41.076 38.08202 ASP O 59.331 30.459 40.305 37.52 203 PRO N 61.473 30.392 40.96639.66 203 PRO CD 62.651 30.883 41.699 40.16 203 PRO CA 61.864 29.41839.934 40.91 203 PRO CB 63.353 29.233 40.180 40.99 203 PRO CG 63.77330.564 40.727 40.90 203 PRO C 61.101 28.123 40.180 42.10 203 PRO O60.710 27.840 41.313 42.51 204 ASP N 60.873 27.334 39.141 43.15 204 ASPCA 60.152 26.079 39.338 44.22 204 ASP CB 59.428 25.647 38.064 44.53 204ASP CG 60.385 25.360 36.925 44.89 204 ASP OD1 61.006 26.325 36.426 45.32204 ASP OD2 60.525 24.179 36.531 45.28 204 ASP C 61.085 24.958 39.76844.93 204 ASP O 62.308 25.133 39.868 44.76 205 GLY N 60.486 23.79840.024 45.69 205 GLY CA 61.263 22.645 40.432 46.42 205 GLY C 61.90622.881 41.783 46.99 205 GLY O 61.328 23.565 42.633 47.23 206 LEU N63.097 22.313 41.980 47.22 206 LEU CA 63.837 22.458 43.235 47.38 206 LEUCB 64.500 21.129 43.653 47.47 206 LEU CG 64.965 21.114 45.126 47.66 206LEU CD1 63.767 21.238 46.078 47.96 206 LEU CD2 65.719 19.822 45.45147.67 206 LEU C 64.914 23.540 43.111 47.44 206 LEU O 65.730 23.55542.179 47.57 207 ALA N 64.909 24.449 44.070 47.44 207 ALA CA 65.86325.536 44.081 47.34 207 ALA CB 65.447 26.596 43.058 47.44 207 ALA C65.849 26.111 45.484 47.38 207 ALA O 65.042 25.715 46.322 47.46 208 GLUN 66.755 27.037 45.749 47.42 208 GLU CA 66.827 27.672 47.056 47.30 208GLU CB 68.024 28.621 47.062 48.01 208 GLU CG 68.202 29.500 48.286 49.08208 GLU CD 69.296 30.532 48.053 49.75 208 GLU OE1 69.087 31.437 47.20950.27 208 GLU OE2 70.370 30.430 48.693 50.06 208 GLU C 65.518 28.43747.308 46.98 208 GLU O 65.086 28.599 48.448 46.85 209 CYS N 64.88628.885 46.225 46.21 209 CYS CA 63.641 29.642 46.305 45.87 209 CYS C62.385 28.832 46.015 46.10 209 CYS O 61.297 29.393 45.886 45.97 209 CYSCB 63.704 30.828 45.344 44.89 209 CYS SG 64.959 32.048 45.809 43.60 210ALA N 62.540 27.517 45.908 46.44 210 ALA CA 61.421 26.623 45.644 47.08210 ALA CB 61.212 26.467 44.142 47.11 210 ALA C 61.720 25.269 46.27747.86 210 ALA O 61.906 24.274 45.581 47.67 211 PRO N 61.766 25.21947.620 48.58 211 PRO CD 61.587 26.356 48.540 48.62 211 PRO CA 62.04823.988 48.366 49.20 211 PRO CB 61.955 24.439 49.823 49.08 211 PRO CG62.341 25.897 49.760 48.69 211 PRO C 61.083 22.849 48.054 50.05 211 PROO 60.004 23.048 47.496 49.93 212 ASN N 61.503 21.642 48.407 51.18 212ASN CA 60.706 20.439 48.206 52.30 212 ASN CB 61.640 19.224 48.139 52.92212 ASN CG 62.555 19.117 49.364 53.44 212 ASN OD1 62.104 18.809 50.47154.23 212 ASN ND2 63.844 19.382 49.169 53.84 212 ASN C 59.773 20.31249.416 52.71 212 ASN O 59.964 20.993 50.424 52.73 213 GLU N 58.78319.432 49.328 53.24 213 GLU CA 57.855 19.238 50.442 53.82 213 GLU CB56.958 18.033 50.171 54.25 213 GLU CG 55.856 18.290 49.155 54.86 213 GLUCD 54.935 17.087 40.983 55.49 213 GLU OE1 55.417 16.037 48.489 55.75 213GLU OE2 53.735 17.182 49.344 55.69 213 GLU C 58.534 19.065 51.813 53.91213 GLU O 58.130 19.704 52.798 54.01 214 ASP N 59.551 18.202 51.88953.72 214 ASP CA 60.255 17.960 53.159 53.41 214 ASP CB 61.297 16.84753.000 54.15 214 ASP CG 62.163 16.644 54.235 54.73 214 ASP OD1 61.67816.044 55.225 55.16 214 ASP OD2 63.341 17.071 54.202 55.21 214 ASP C60.936 19.189 53.736 52.79 214 ASP O 61.118 19.282 54.953 52.99 215 GLNN 61.335 20.122 52.880 51.88 215 GLN CA 61.974 21.331 53.383 50.96 215GLN CB 62.968 21.910 52.366 51.60 215 GLN CG 63.931 22.928 52.975 52.22215 GLN CD 65.005 22.267 53.828 52.81 215 GLN OE1 64.703 21.457 54.71453.24 215 GLN NE2 66.268 22.605 53.563 52.88 215 GLN C 60.888 22.36353.685 49.85 215 GLN O 61.183 23.472 54.140 49.74 216 GLY N 59.63521.989 53.414 48.46 216 GLY CA 58.515 22.880 53.679 46.62 216 GLY C57.625 23.281 52.506 45.32 216 GLY O 56.666 24.037 52.692 44.87 217 GLYN 57.928 22.793 51.303 43.79 217 GLY CA 57.107 23.139 50.151 42.16 217GLY C 57.490 24.463 49.506 40.85 217 GLY O 58.503 25.064 49.856 40.57218 LYS N 56.675 24.928 48.566 39.66 218 LYS CA 56.962 26.180 47.86938.74 218 LYS CB 56.289 26.165 46.488 38.96 218 LYS CG 56.559 24.90745.658 39.24 218 LYS CD 58.053 24.587 45.548 39.71 218 LYS CE 58.28323.288 44.756 39.75 218 LYS NZ 59.659 22.715 44.945 40.80 218 LYS C56.540 27.456 48.627 37.69 218 LYS O 55.681 27.412 49.504 37.33 219 TYRN 57.171 28.579 48.280 36.44 219 TYR CA 56.861 29.880 48.871 35.08 219TYR CB 58.081 30.790 48.826 35.14 219 TYR CG 59.141 30.373 49.811 35.36219 TYR CD1 58.947 30.542 51.184 35.32 219 TYR CE1 59.897 30.095 52.11135.38 219 TYR CD2 60.314 29.749 49.380 35.55 219 TYR CE2 61.269 29.29950.294 35.73 219 TYR CZ 61.055 29.475 51.655 35.85 219 TYR OH 62.01229.037 52.552 35.93 219 TYR C 55.687 30.502 48.117 34.52 219 TYR O55.322 30.036 47.035 33.65 220 VAL N 55.103 31.552 48.689 33.96 220 VALCA 53.916 32.183 48.114 33.59 220 VAL CB 53.419 33.356 49.003 33.34 220VAL CG1 54.312 34.585 48.815 32.91 220 VAL CG2 51.963 33.673 48.65932.96 220 VAL C 53.999 32.657 46.665 33.69 220 VAL O 52.982 32.66545.961 33.62 221 MET N 55.189 33.045 46.211 33.66 221 MET CA 55.33833.499 44.827 34.39 221 MET CB 56.464 34.536 44.698 33.60 221 MET CG56.167 35.877 45.391 33.08 221 MET SD 54.466 36.501 45.157 32.05 221 METCE 54.422 36.801 43.385 32.38 221 MET C 55.548 32.371 43.811 35.23 221MET O 55.907 32.626 42.659 35.28 222 TYR N 55.345 31.126 44.232 35.97222 TYR CA 55.463 30.014 43.298 36.93 222 TYR CB 55.432 28.673 44.02737.59 222 TYR CG 55.974 27.540 43.187 38.08 222 TYR CD1 57.327 27.47842.866 38.44 222 TYR CE1 57.832 26.463 42.052 38.71 222 TYR CD2 55.12926.554 42.678 38.39 222 TYR CE2 55.621 25.533 41.868 38.73 222 TYR CZ56.976 25.498 41.559 39.11 222 TYR OH 57.471 24.499 40.746 40.00 222 TYRC 54.219 30.169 42.414 37.37 222 TYR O 53.140 30.513 42.902 37.08 223PRO N 54.354 29.920 41.103 38.04 223 PRO CD 55.614 29.594 40.418 38.08223 PRO CA 53.256 30.042 40.135 38.39 223 PRO CB 53.979 29.955 38.79638.48 223 PRO CG 55.112 29.055 39.095 38.40 223 PRO C 52.079 29.07740.200 38.94 223 PRO O 50.947 29.448 39.890 38.98 224 ILE N 52.32327.847 40.618 39.26 224 ILE CA 51.244 26.886 40.628 40.09 224 ILE CB51.400 25.917 39.451 40.48 224 ILE CG2 50.837 26.567 38.185 40.60 224ILE CG1 52.880 25.526 39.317 40.80 224 ILE CD1 53.147 24.535 38.21141.41 224 ILE C 51.056 26.058 41.873 40.24 224 ILE O 51.989 25.82142.638 40.30 225 ALA N 49.816 25.614 42.034 40.58 225 ALA CA 49.38024.764 43.127 40.97 225 ALA CB 49.722 23.308 42.795 41.20 225 ALA C49.825 25.070 44.556 41.21 225 ALA O 49.990 24.139 45.343 41.39 226 VALN 50.037 26.330 44.920 41.25 226 VAL CA 50.394 26.568 46.314 41.45 226VAL CB 51.272 27.840 46.493 41.51 226 VAL CG1 51.068 28.797 45.334 41.64226 VAL CG2 50.969 28.515 47.827 41.44 226 VAL C 49.057 26.630 47.07241.43 226 VAL O 48.406 27.668 47.171 41.19 227 SER N 48.652 25.46047.564 41.45 227 SER CA 47.397 25.236 48.289 41.89 227 SER CB 47.13223.736 48.353 41.88 227 SER OG 48.230 23.085 48.982 41.75 227 SER C47.298 25.775 49.708 41.96 227 SER O 46.190 25.967 50.228 42.00 228 GLYN 48.447 25.990 50.337 41.99 228 GLY CA 48.466 26.463 51.709 42.37 228GLY C 48.642 25.275 52.635 42.72 228 GLY O 48.721 25.426 53.853 42.58229 ASP N 48.710 24.087 52.037 43.09 229 ASP CA 48.874 22.835 52.77243.59 229 ASP CB 48.539 21.636 51.884 44.44 229 ASP CG 47.071 21.55851.518 45.07 229 ASP OD1 46.698 20.559 50.865 46.12 229 ASP OD2 46.29422.472 51.863 45.27 229 ASP C 50.287 22.645 53.291 43.55 229 ASP O50.550 21.722 54.060 43.45 230 HIS N 51.197 23.512 52.865 43.38 230 HISCA 52.586 23.411 53.287 43.24 230 HIS CB 53.455 23.095 52.076 43.74 230HIS CG 53.018 21.863 51.355 44.30 230 HIS CD2 52.116 21.690 50.357 44.53230 HIS ND1 53.423 20.601 51.728 44.35 230 HIS CE1 52.787 19.703 50.99744.69 230 HIS NE2 51.988 20.339 50.158 44.94 230 HIS C 53.064 24.67353.975 42.89 230 HIS O 52.631 25.780 53.637 42.41 231 GLU N 53.96324.484 54.939 42.49 231 GLU CA 54.511 25.574 55.735 42.28 231 GLU CB55.656 25.064 56.625 43.29 231 GLU CG 56.270 26.150 57.523 44.54 231 GLUCD 57.300 25.603 58.521 45.36 231 GLU OE1 57.909 26.418 59.265 45.54 231GLU OE2 57.492 24.365 58.558 45.66 231 GLU C 55.001 26.786 54.957 41.71231 GLU O 54.627 27.915 55.277 41.40 232 ASN N 55.833 26.563 53.94140.80 232 ASN CA 56.381 27.684 53.184 40.30 232 ASN CB 57.548 27.22852.289 40.22 232 ASN CG 58.721 26.673 53.086 40.58 232 ASN OD1 59.05327.175 54.157 40.38 232 ASN ND2 59.364 25.642 52.550 40.48 232 ASN C55.359 28.434 52.336 39.58 232 ASN O 55.616 29.562 51.928 39.26 233 ASNN 54.215 27.815 52.073 39.33 233 ASN CA 53.182 28.447 51.252 39.40 233ASN CB 51.904 27.605 51.239 39.00 233 ASN CG 52.081 26.256 50.543 38.74233 ASN OD1 51.163 25.441 50.523 38.17 233 ASN ND2 53.259 26.021 49.97338.66 233 ASN C 52.832 29.863 51.702 39.53 233 ASN O 52.547 30.71950.871 39.63 234 LYS N 52.867 30.101 53.012 39.81 234 LYS CA 52.53231.403 53.594 40.33 234 LYS CB 51.901 31.226 54.978 40.90 234 LYS CG50.714 30.297 55.069 41.90 234 LYS CD 50.448 30.006 56.544 42.73 234 LYSCE 49.282 29.054 56.742 43.39 234 LYS NZ 49.502 27.769 56.016 44.13 234LYS C 53.708 32.361 53.772 40.21 234 LYS O 53.569 33.363 54.484 40.40235 MET N 54.860 32.066 53.173 39.47 235 MET CA 56.014 32.946 53.33339.09 235 MET CB 57.052 32.289 54.244 40.17 235 MET CG 56.465 31.68955.512 41.84 235 MET SD 57.740 30.990 56.579 44.64 235 MET CE 58.08329.387 55.761 43.33 235 MET C 56.674 33.312 52.013 38.33 235 MET O56.446 32.657 50.988 37.96 236 PHE N 57.490 34.360 52.046 37.26 236 PHECA 58.204 34.808 50.859 37.15 236 PHE CB 58.372 36.326 50.881 36.44 236PHE CG 57.125 37.077 50.504 35.82 236 PHE CD1 56.721 37.153 49.172 35.71236 PHE CD2 56.344 37.686 51.477 35.44 236 PHE CE1 55.554 37.832 48.81635.24 236 PHE CE2 55.174 38.367 51.132 35.20 236 PHE CZ 54.777 38.43949.800 35.07 236 PHE C 59.577 34.146 50.763 37.28 236 PHE O 60.24333.922 51.770 36.90 237 SER N 59.987 33.823 49.541 37.66 237 SER CA61.284 33.205 49.312 38.05 237 SER CB 61.321 32.542 47.935 38.00 237 SEROG 61.120 33.505 46.917 37.41 237 SER C 62.342 34.293 49.357 38.70 237SER O 62.024 35.486 49.375 38.35 238 GLN N 63.601 33.869 49.379 39.50238 GLN CA 64.726 34.790 49.380 40.45 238 GLN CB 66.048 24.010 49.45841.34 238 GLN CG 66.243 33.277 50.783 42.67 238 GLN CD 66.412 34.24251.951 43.54 238 GLN OE1 66.160 33.891 53.111 44.24 238 GLN NE2 66.84935.468 51.649 43.86 238 GLN C 64.671 35.590 48.085 40.43 238 GLN O65.017 36.769 48.059 40.66 239 CYS N 64.225 34.939 47.013 40.63 239 CYSCA 64.120 35.586 45.703 40.74 239 CYS C 63.102 36.727 45.725 40.41 239CYS O 63.356 37.808 45.184 40.04 239 CYS CB 63.739 34.559 44.628 41.45239 CYS SG 65.042 33.335 44.236 42.69 240 SER N 61.954 36.479 46.34940.19 240 SER CA 60.910 37.493 46.462 40.29 240 SER CB 59.643 36.88247.071 40.08 240 SER OG 59.074 35.910 46.215 39.76 240 SER C 61.38338.659 47.335 40.40 240 SER O 61.078 39.823 47.053 40.33 241 LYS N62.127 38.339 48.391 40.50 241 LYS CA 62.637 39.354 49.308 41.10 241 LYSCB 63.365 38.698 50.481 40.76 241 LYS CG 62.460 38.012 51.491 40.60 241LYS CD 63.282 37.429 52.628 40.39 241 LYS CE 62.395 36.840 53.707 40.49241 LYS NZ 63.204 36.274 54.818 40.45 241 LYS C 63.586 40.329 48.62041.77 241 LYS O 63.494 41.544 48.810 41.57 242 GLN N 64.505 39.78847.826 42.60 242 GLN CA 65.470 40.613 47.113 43.55 242 GLN CB 66.39839.712 46.281 44.45 242 GLN CG 67.611 40.417 45.716 45.86 242 GLN CD68.577 39.463 45.038 46.74 242 GLN OE1 68.199 38.719 44.133 47.71 242GLN NE2 69.834 39.486 45.467 47.24 242 GLN C 64.731 41.607 46.209 43.65242 GLN O 65.047 42.797 46.170 43.59 243 SER N 63.734 41.113 45.48543.77 243 SER CA 62.961 41.959 44.588 43.87 243 SER CB 62.028 41.09243.740 43.76 243 SER OG 62.771 40.173 42.961 43.31 243 SER C 62.14543.006 45.337 44.23 243 SER O 62.098 44.171 44.940 44.17 244 ILE N61.510 42.588 46.427 44.68 244 ILE CA 60.668 43.480 47.218 45.21 244 ILECB 59.730 42.656 48.137 44.81 244 ILE CG2 59.051 43.562 49.171 44.62 244ILE CG1 58.717 41.909 47.258 44.59 244 ILE CD1 57.892 40.891 48.01444.05 244 ILE C 61.467 44.499 48.025 46.05 244 ILE O 61.017 45.62748.224 45.65 245 TYR N 62.651 44.102 48.481 47.25 245 TYR CA 63.51145.006 49.233 48.50 245 TYR CB 64.825 44.320 49.611 49.13 245 TYR CG65.755 45.191 50.434 49.90 245 TYR CD1 65.463 45.497 51.766 50.03 245TYR CE1 66.312 46.302 52.526 50.40 245 TYR CD2 66.922 45.715 49.87850.27 245 TYR CE2 67.781 46.527 50.630 50.62 245 TYR CZ 67.471 46.81551.952 50.61 245 TYR OH 68.321 47.612 52.693 50.69 245 TYR C 63.79946.187 48.315 49.10 245 TYR O 63.675 47.341 48.716 49.04 246 LYS N64.174 45.887 47.074 49.81 246 LYS CA 64.452 46.927 46.090 50.75 246 LYSCB 64.673 46.323 44.695 51.00 246 LYS CG 65.982 45.571 44.502 51.59 246LYS CD 66.129 45.125 43.052 51.90 246 LYS CE 67.416 44.357 42.830 52.28246 LYS NZ 67.560 43.948 41.403 52.33 246 LYS C 63.273 47.890 46.02351.14 246 LYS O 63.440 49.103 46.118 51.15 247 THR N 62.078 47.33645.855 51.80 247 THR CA 60.866 48.139 45.773 52.40 247 THR CB 59.61447.248 45.651 52.30 247 THR OG1 59.664 46.514 44.420 52.09 247 THR CG258.346 48.107 45.684 52.13 247 THR C 60.702 49.033 46.998 53.14 247 THRO 60.542 50.246 46.877 53.08 248 ILE N 60.750 48.428 48.176 53.94 248ILE CA 60.583 49.178 49.408 54.96 248 ILE CB 60.655 48.240 50.637 54.88248 ILE CG2 60.528 49.057 51.925 54.90 248 ILE CG1 59.562 47.177 50.52654.89 248 ILE CD1 59.554 46.217 51.686 54.95 248 ILE C 61.593 50.31449.570 55.69 248 ILE O 61.207 51.454 49.833 55.77 249 GLU N 62.87750.027 49.393 56.54 249 GLU CA 63.878 51.071 49.578 57.48 249 GLU CB65.289 50.469 49.652 58.05 249 GLU CG 65.828 49.905 48.351 59.02 249 GLUCD 67.257 49.398 48.493 59.54 249 GLU OE1 68.160 50.222 48.760 59.84 249GLU OE2 67.476 48.175 48.345 60.02 249 GLU C 63.843 52.193 48.543 57.80249 GLU O 64.546 53.192 48.693 57.86 250 SER N 63.025 52.055 47.50558.16 250 SER CA 62.953 53.105 46.494 58.45 250 SER CB 63.514 52.59845.165 58.61 250 SER OG 62.729 51.541 44.647 58.94 250 SER C 61.55453.672 46.269 58.59 250 SER O 61.387 54.623 45.506 58.57 251 LYS N60.554 53.098 46.935 58.76 251 LYS CA 59.172 53.554 46.781 58.85 251 LYSCB 58.351 52.516 46.017 58.99 251 LYS CG 58.596 52.438 44.520 59.31 251LYS CD 57.673 51.377 43.940 59.49 251 LYS CE 57.609 51.388 42.423 59.66251 LYS NZ 56.579 50.404 41.938 59.42 251 LYS C 58.474 53.839 48.10658.77 251 LYS O 57.508 54.597 48.152 58.81 252 ALA N 58.945 53.21749.179 58.69 252 ALA CA 58.339 53.429 50.489 58.69 252 ALA CB 59.16052.721 51.573 58.66 252 ALA C 58.273 54.926 50.772 58.54 252 ALA O57.297 55.431 51.331 58.67 253 GLN N 59.320 55.635 50.364 58.33 253 GLNCA 59.396 57.071 50.575 57.97 253 GLN CB 60.815 57.559 50.278 58.55 253GLN CG 61.072 59.009 50.627 59.13 253 GLN CD 60.856 59.299 52.101 59.51253 GLN OE1 59.718 59.354 52.577 59.79 253 GLN NE2 61.952 59.480 52.83559.76 253 GLN C 58.399 57.788 49.676 57.38 253 GLN O 57.889 58.85650.024 57.51 254 GLU N 58.114 57.180 48.528 56.50 254 GLU CA 57.19357.747 47.551 55.63 254 GLU CB 57.363 57.039 46.204 56.07 254 GLU CG56.591 57.692 45.060 56.47 254 GLU CD 56.739 56.943 43.746 56.78 254 GLUOE1 57.891 56.701 43.318 57.11 254 GLU OE2 55.702 56.602 43.139 56.77254 GLU C 55.709 57.735 47.941 54.80 254 GLU O 55.030 58.753 47.80154.58 255 CYS N 55.195 56.602 48.422 53.66 255 CYS CA 53.778 56.54748.783 52.59 255 CYS C 53.410 55.742 50.023 52.52 255 CYS O 52.23155.633 50.345 52.30 255 CYS CB 52.954 56.028 47.603 51.44 255 CYS SG53.117 54.239 47.304 49.73 256 PHE N 54.393 55.171 50.712 52.58 256 PHECA 54.095 54.408 51.922 52.90 256 PHE CB 55.255 53.479 52.288 52.57 256PHE CG 55.233 52.163 51.570 52.17 256 PHE CD1 54.791 52.079 50.252 52.00256 PHE CD2 55.701 51.013 52.194 51.99 256 PHE CE1 54.819 50.872 49.56651.86 256 PHE CE2 55.733 49.797 51.512 51.86 256 PHE CZ 55.291 49.72850.196 51.82 256 PHE C 53.832 55.376 53.066 53.32 256 PHE O 54.47356.421 53.164 53.19 257 GLN N 52.888 55.022 53.930 53.81 257 GLN CA52.529 55.874 55.057 54.49 257 GLN CB 51.075 56.327 54.924 54.41 257 GLNCG 50.729 56.968 53.593 54.72 257 GLN CD 49.269 57.361 53.509 54.80 257GLN OE1 48.826 57.946 52.521 55.05 257 GLN NE2 48.510 57.038 54.54954.80 257 GLN C 52.683 55.125 56.369 54.95 257 GLN O 53.133 53.98156.396 55.09 258 GLU N 52.309 55.788 57.458 55.42 258 GLU CA 52.35355.184 58.781 55.77 258 GLU CB 52.566 56.261 59.851 56.14 258 GLU CG53.849 57.070 59.678 56.74 258 GLU CD 54.055 58.095 60.779 57.19 258 GLUOE1 55.140 58.729 60.819 57.35 258 GLU OE2 53.129 58.270 61.606 57.42258 GLU C 50.973 54.554 58.949 55.81 258 GLU O 50.057 54.864 58.18555.77 259 ARG N 50.814 53.664 59.921 55.84 259 ARG CA 49.506 53.05860.133 55.95 259 ARG CB 49.577 51.980 61.212 55.87 259 ARG CG 50.09450.635 60.726 55.84 259 ARG CD 50.023 49.614 61.849 55.69 259 ARG NE50.405 40.261 61.442 55.63 259 ARG CZ 49.673 47.461 60.670 55.58 259 ARGNH1 48.498 47.860 60.196 55.45 259 ARG NH2 50.113 46.245 60.387 55.58259 ARG C 48.504 54.143 60.542 56.13 259 ARG O 47.427 54.231 59.91156.19 259 ARG OXT 48.802 54.904 61.491 56.12 260 007 N3 47.727 29.82939.980 39.58 260 007 O6 47.576 27.266 41.351 41.05 260 007 O1 44.78823.897 40.843 43.59 260 007 C5 47.174 31.040 40.173 38.64 260 007 O249.740 36.283 39.759 35.36 260 007 C1 46.829 28.736 39.597 40.68 260 007C2 47.378 28.034 38.342 40.79 260 007 C3 47.601 29.053 37.224 40.76 260007 C4 48.428 28.495 36.081 40.93 260 007 C6 46.620 27.701 40.709 41.02260 007 C7 45.036 26.131 41.645 42.36 260 007 C8 44.100 26.443 42.82342.38 260 007 C13 44.799 27.056 44.045 42.39 260 007 C20 46.068 26.28244.406 42.54 260 007 C21 43.850 27.067 45.249 42.60 260 007 N1 48.73435.382 39.482 36.20 260 007 C9 49.235 34.151 39.306 36.48 260 007 O350.444 33.937 39.245 35.65 260 007 C10 48.255 33.016 39.175 37.20 260007 C22 48.177 32.158 40.439 38.16 260 007 C23 47.749 32.989 41.66537.95 260 007 O4 45.975 31.286 40.126 38.63 260 007 N2 45.330 27.35540.904 41.60 260 007 C11 44.380 25.075 40.766 42.75 260 007 O5 43.45325.430 40.003 43.03 260 007 C14 47.884 32.183 42.942 38.16 260 007 C1549.090 32.237 43.694 38.38 260 007 C16 49.209 31.514 44.917 38.20 260007 C17 48.120 30.724 45.385 38.22 260 007 C18 46.921 30.645 44.62338.22 260 007 C19 46.801 31.375 43.401 38.18 260 007 C12 46.392 26.94237.907 41.12 261 ZN2 ZN + 2 51.599 35.407 39.845 36.08

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which arehereby incorporated by reference in their entireties.

1. An isolated polypeptide comprising the amino acid sequence of SEQ IDNO: 8 wherein residue 139 is a member selected from the group consistingof A, R, N, D, C, E, Q, G, H, K, P, S, T and Y.
 2. A chimeric proteincomprising the polypeptide of claim
 1. 3. The polypeptide of claim 1wherein the amino acid residue at position 139 of SEQ ID NO: 8 isselected from the group consisting alanine, glycine and serine.
 4. Anisolated polypeptide of claim 1 wherein said polypeptide: (a) catalyzesthe proteolytic cleavage of a polypeptide comprising the amino acidsequence of SEQ ID NO: 17; and (b) binds toN-(D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl)-L-3-amino-2-dimethylbutanoyl-L-alanine, 2-(amino)ethyl amide;(4-biphenylsulfonyl)amino-propionic acid3,4-dimethyl-2-(N-3′-pyridylmethyl-p-methoxysulfonamido)-benzenehydroxyamicacid; alpha(R)-[[[4-2-butynyloxy) phenyl]sulfonyl]amino]benzenepropanoicacid; N-hydroxy-alpha(R),3(S)-dimethyl-3-[4-[(2-methyl-4-quinolinyl)methoxy]phenyl]-2-oxo-1-pyrrolidineacetamide;or N-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH.
 5. Achimeric protein comprising the polypeptide of claim
 4. 6. A crystalcomprising a polypeptide comprising the amino acid sequence of SEQ IDNO: 20 that has the space group P2₁2₁2₁, with unit cell dimensions ofa=73, b=75, c=103 Angstroms.
 7. The crystal of claim 6 wherein saidpolypeptide is complexed with a ligand which is a member selected fromthe group consisting ofN-[D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl)-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide; (4-biphenylsulfonyl)amino-propionic acid3,4-dimethyl-2-(N-3′-pyridylmethyl-p-methoxysulfonamido)-benzenehydroxyamicacid; alpha(R)-[[[4-2-butynyloxy) phenyl]sulfonyl]amino]benzenepropanoicacid;N-hydroxy-alpha(R),3(S)-dimethyl-3-[4-[(2-methyl-4-quinolinyl)methoxy]phenyl]-2-oxo-1-pyrrolidineacetamide;and N-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH. 8.The crystal of claim 7 wherein the ligand is[N-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH andwherein the crystal is characterized by the structural coordinates setforth in Table
 3. 9. The polypeptide of claim 4 complexed with a memberselected from the group consisting of a polypeptide comprising the aminoacid sequence of SEQ ID NO: 17;N-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-L-3-amino-2-dimethylbutanoyl-L-alanine,2-(amino)ethyl amide; (4-biphenylsulfonyl)amino-propionic acid3,4-dimethyl-2-(N-3′-pyridylmethyl-p-methoxysulfonamido)-benzenehydroxyamicacid; alpha(R)-[[[4-2-butynyloxy) phenyl]sulfonyl]amino]benzenepropanoicacid;N-hydroxy-alpha(R),3(S)-dimethyl-3-[4-[(2-methyl-4-quinolinyl)methoxy]phenyl]-2-oxo-1-pyrrolidineacetamide;and N-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH. 10.The polypeptide of claim 1 comprising the amino acid sequence of SEQ IDNO:
 20. 11. A crystal comprising a polypeptide comprising the amino acidsequence of SEQ ID NO: 20 complexed withN-{3-(hydroxyaminocarbonyl)-1-oxo-(2R)-benzylpropyl}-Ile-Leu-OH that hasthe space group P2₁2₁2₁, with unit cell dimensions of a73, b=75, c=103Angstroms.